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PHYSIOLOGICAL    CHEMISTRY 

LONG 


Digitized  by  tlie  Internet  Arcliive 

in  2010  witli  funding  from 

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http://www.archive.org/details/textbookofphysioOOIong 


A  TEXT-BOOK 


OF 


PHYSIOLOGICAL  CHEMISTRY 


FOR 


STUDENTS   OF  MEDICINE 


BY 


JOHN    H.  LONG,   M.S.,   Sc.D. 

PROFESSOR   OF   CHEMISTRY   IN   NORTHWESTERN   UNIVERSITY   MEDICAL   SCHOOL,   CHICAGO 


irUustrateD 


PHILADELPHIA 

P.    BLAKISfON'S   SON    &    CO 

IOI2  Walnut  Street 

1905 


Copyright,  1905,  by  P.  Blakiston's  Son  &  Co. 


Press  of 

The  New  Era  Psinting  Compahy. 

Lahcasieii,  Pjk 


PREFACE. 

In  the  following  pages  I  have  attempted  to  present  a  brief 
account  of  the  important  principles  of  physiological  chemistry 
in  a  form  suitable  for  the  use  of  medical  students  who  may  be 
assumed  to  have  completed  courses  in  the  elements  of  general 
inorganic  and  organic  chemistry.  From  the  very  necessities 
of  the  case  a  work  of  this  character,  dealing  with  many  topics 
in  an  elementary  way,  must  be  largely  a  compilation ;  in  the 
selection  of  material,  besides  consulting  the  standard  hand 
books,  I  have  made  free  use  of  the  recent  monographs  by 
Cohnheim,  Effront  and  Oppenheimer,  as  well  as  of  numerous 
articles  in  the  Zeitschrift  fiir  physiologische  Chemie,  the 
Beitrage  zur  chemischen  Physiologic  und  Pathologic  and  other 
journals.  As  the  book  is  intended  for  beginners  I  have  not 
thought  it  necessary  to  make  any  special  quotations  of  litera- 
ture references. 

In  addition  to  the  usual  topics  of  discussion  in  elementary 
works  on  physiological  chemistry  I  have  given  an  outline  of 
the  chemical  phases  of  the  recent  theories  of  immunity,  and 
a  short  explanation  of  the  important  applications  of  the 
methods  of  cr}^oscopy  and  electrical  conductivity,  and  other 
physical  processes,  in  the  field  of  chemistry  related  to  medi- 
cine. Work  in  these  latter  lines  must  soon  become  a  part  of 
the  laboratory  training  of  medical  students. 

A  considerable  number  of  illustrative  experiments  are  given 
in  the  text,  but  distinguished  by  being  printed  in  smaller  type. 
These  experiments  are  sufficiently  numerous  and  comprehen- 
sive to  serve  the  purpose  of  a  laboratory  course  parallel  with 
the  general  course. 

In  the  work  of  proofreading  I  have  received  very  material 

assistance  from  my  colleague.  Dr.  W.  H.  Buhlig.  to  whom 

my  sincere  thanks  are  due. 

J.   H.  Long. 
Chicago,  July,  1905. 


TABLE  OF  CONTENTS. 


INTRODUCTION 
Chapter  I.  Scope  and  Methods i 

SECTION    I 
THE    NUTRITIVES 

Chapter           II.  Inorganic  Elements.    Water.    Air.     Salts  9 
Chapter         III.  The    Carbohydrates    and    Related    Sub- 
stances     22 

Chapter         IV.  The  Fats  and  Substances  Related  to  Them  51 

Chapter           V.  The   Protein   Substances 64 

SECTION    II 
FERMENTS    AND    DIGESTIVE   PROCESSES 

Chapter  VI.  Enzymes  and  Other  Ferments.    Digestion  115 

Chapter       VII.  Saliva  and  Salivary  Digestion 144 

Chapter      VIII.  The  Gastric  Juice  and  Changes  in  the 

Stomach   150 

Chapter         IX.  The  Products  of  Pancreatic  Digestion.  .  .    173 
Chapter  X.  Changes  in  the  Intestines.     Feces 192 

SECTION    III 

THE   CHEMISTRY  OF  THE  BLOOD,   THE  TISSUES   AND 
SECRETIONS    OF   THE    BODY 

Chapter         XL  The  Blood   213 

Chapter  XII.  The  Optical  Properties  of  Blood.  The 
Use  of  the  Spectroscope  and  Other 
Instruments   234 

Chapter      XIII.  Further  Physical  Methods  in  Blood  Ex- 
amination.    Freezing    Point   and   Elec- 
trical Conductivity.      The  Hematocrit.  .   248 
vii 


VUl  TABLE    OF    CONTENTS. 

Chapter  XIV.  Sonic  Special  Properties  of  Blood 
Serum.  Bactericidal  Action.  Precipi- 
tins. Agip^lutinins.  Ijacteriolysins.  He- 
molysins      264 

Chapter        XV.  Transudations  Related  to  the  Blood 280 

Chapter      XVI.  Milk   286 

Chapter     XVII.  The  Chemistry  of  the  Liver.      Bile.     Cells 

in  General   302 

Chapter  X\TII.  Chemistry  of  the  Pancreas  and  Other 
Glands.  Muscle,  Bone,  the  Hair  and 
Other   Tissues    329 

SECTION    IV 

THE  END  PRODUCTS  OF  METABOLISM.  EXCRETIONS. 
ENERGY  BALANCE 

Chapter      XIX.  The  Nitrogenous  Excretion.    Urine 351 

Chapter        XX.  The  Gaseous  Excretion.    Respiration ....  380 

Chapter      XXI.  The  Energy  Equation 392 

Index    411 


PHYSIOLOGICAL   CHEMISTRY. 


INTRODUCTION. 

CHAPTER    I. 

Scope  and  Methods.  In  our  study  of  the  organized  world 
the  most  fundamental  problems  which  present  themselves  are 
essentially  chemical.  Beginning  with  the  mysterious  trans- 
formations wrought  through  the  energy  of  the  sun's  rays  in 
such  simple  substances  as  the  carbon  dioxide  and  aqueous 
vapor  of  the  atmosphere,  when  these  bodies  come  in  contact 
within  certain  vegetable  cells,  and  following  the  history  of  the 
products  thus  formed  through  their  many  changes  in  the 
plant  organism  and  later  through  the  highly  complex  animal 
structures,  for  whose  formation  the  plant  cell  must  prepare 
the  raw  material,  and  finally  as  we  note  the  gradual  breaking 
clown  of  these  same  elaborate  combinations,  with  liberation 
of  energy  and  ultimate  restoration  of  carbon  dioxide  and 
water  and  nitrogen  to  the  air  and  soil  which  once  had  held 
them,  we  see  that,  step  by  step,  the  various  transformations 
which  occur  are  such  as  may  be  represented  by  the  equations 
of  organic  chemistry.  It  may  not  always  be  possible  to 
express  these  equations  in  simple  or  exact  form,  because  of 
the  lack  of  knowledge  in  details,  but  the  theoretical  feasi- 
bility of  writing  such  expressions  we  everywhere  recognize. 

In  following  the  migrations  of  atoms  of  carbon,  hydrogen, 
oxygen  and  nitrogen  through  the  vegetable  and  animal  worlds, 
our  inquiry  naturally  widens  beyond  the  field  legitimately 
claimed  by  chemistry.  We  find  ourselves  at  the  very  outset 
confronted  by  the  question  of  the  final  forces  inaugurating 


2  PHYSIOLOGICAL    CHEMISTRY. 

the  changes,  the  chemical  expression  of  Avhich  appears  often 
so  extremely  simple.  It  may  be  the  part  of  wisdom  to  admit 
at  once  that  this  cjiiestion  is  one  beyond  our  power  to  answer. 
Then,  again,  we  find  ourselves  attracted  by  questions  of  form, 
function  and  general  conditions  of  the  existence  of  organisms 
upon  the  earth  in  addition  to  those  of  composition  and  mode 
of  formation.  In  touching  these  we  enter  upon  the  field  of 
General  Biology^  and  soon  recognize  that  in  this  vast  and  inde- 
pendent science  there  is  much  contained  which  has  no  possible 
bearing  on  our  problems  of  chemistry.  But  some  knowledge 
of  biological  science  is  certainly  essential  to  a  proper  under- 
standing of  the  chemistry  of  living  beings. 

As  proper  subjects  of  inquiry  in  Physiological  Chemistry 
we  recognize  mainly  the  following:  (a)  The  nutrition  of 
plants  and  animals  and  the  composition  and  properties  of 
the  nutrient  substances,  (b)  The  changes  which  the  nutrients 
undergo  before  and  during  the  processes  of  assimilation,  (c) 
The  agents  of  preparation  for  assimilation  and  the  general 
conditions  of  their  activity,  (d)  The  fate  of  the  assimilated 
nutrients  and  the  nature  of  the  products  of  degradation,  (c) 
The  absorption  or  liberation  of  energy. 

In  the  broader  sense  the  discussion  is  extended  to  the  con- 
ditions appearing  in  the  life  history  of  plants  and  the  lower 
animals  as  well  as  of  man,  but  ordinarily  the  narrower  field 
of  the  life  of  man  and  the  higher  animals  alone  is  considered, 
and  in  the  present  work  the  latter  limits  will  be  observed.  But 
a  brief  discussion  of  the  general  relations  of  plants  to  animals 
will  not  be  out  of  place. 

At  one  time  it  was  very  generally  held  that  the  cell  activ- 
ities in  the  plant  are  essentially  different  from  those  in  the 
animal,  the  work  in  the  one  case  being  looked  upon  as  wholly 
synthetic,  while  in  the  other  it  was  assumed  to  be  disinte- 
gration or  analysis.  But  this  is  not  quite  correct.  The  reac- 
tions in  the  two  classes  of  structures  are  qualitatively  much 
the  same,  although  the  quantitative  differences  are  so  great 
that  -we  almost   lose   sight   of   qualitative   similarities.     The 


INTRODUCTION.  3 

reactions  in  the  plant  world  are  largely  endothermal  and 
require  for  their  completion  the  constant  expenditure  of  ex- 
ternal energy.  This  energy  is  derived  from  sunlight  and 
through  its  agency  chlorophyll-bearing  plants  are  able  to  effect 
a  remarkable  condensation,  viz. :  that  of  carbon  dioxide  with 
water  accompanied  by  liberation  of  oxygen.  In  its  simplest 
terms  this  condensation  may  be  represented  as 

C02-)-H20  =  H2CO  +  02; 

that  is,  formaldehyde  and  oxygen  result. 

Formaldehyde  is  the  first  member  of  the  series  containing 
the  monosaccharoses  and  may  be  the  actual  starting  point  in 
their  elaboration  by  the  vegetable  cell.  Of  the  mechanism  of 
further  transformations  by  the  plant  we  know  but  little;  it  is 
likely  that  many  of  the  following  changes  are  brought  about 
by  the  action  of  soluble  ferments  or  enzymes,  which  will  be 
referred  to  in  a  subsequent  chapter.  With  the  completion  of 
this  synthesis  a  large  amount  of  kinetic  energy  of  the  solar 
rays  is  transformed  into  the  potential  energy  of  protein,  fat 
or  carbohydrate.  In  the  oxidation  of  the  plant  as  fuel  or 
food  the  opposite  change  is  accomplished,  and  the  stored  up 
energy  in  complex  organic  molecules  is  liberated  as  heat,  elec- 
tricity or  muscular  motion.  These  reactions  are  so  charac- 
teristic for  plants  and  animals  that  Ave  are  apt  to  lose  sight  of 
others  which  also  take  place.  In  plants  there  is  a  respiration 
process  as  in  animals,  in  which  oxygen  is  absorbed  and  carbon 
dioxide  liberated,  and  in  the  dark  this  may  be  readily  observed, 
since  then  it  is  not  obscured  by  the  much  more  prominent  re- 
duction process.  Indeed,  this  respiration  may  be  followed  in  the 
light  in  the  case  of  those  plants  which  are  free  from  chloro- 
phyll. Further  than  this  there  are  parasitic  plants  which,  free 
from  chlorophyll,  must  depend  on  other  plants  for  their  nour- 
ishment; they  consume  organic  and  not  inorganic  materials, 
and  in  this  behavior  resemble  animals  completely. 

Then  it  must  be  remembered  that  the  activity  in  the  animal 
is  not  wholly  oxidation  or  degradation.     It  is  well  known  that 


4  PHYSIOLOGICAL    CHEMISTRY. 

some  syntheses  are  constantly  taking  place  which  are  com- 
monly overlooked  because  of  the  much  greater  importance  of 
the  oxidation  reactions.  In  recent  years  low  forms  of  animal 
life  have  been  found  which  contain  chlorophyll  grains  and 
which  are  able  to  produce  oxygen  in  presence  of  sunlight.  In 
some  of  these  cases  the  chlorophyll  may  be  present  in  a  sym- 
biont  organism,  but  in  others  it  appears  to  be  diffuse,  and 
therefore  brings  the  animal  structure  containing  it  into  close 
relation  with  vegetable  cells. 

In  the  essential  phenomena  of  life  plants  and  animals  have, 
then,  much  in  common ;  it  is  onh'  \\-hen  we  follo\\'  them  into 
details  that  the  characteristic  differences  appear. 

From  the  nature  of  the  materials  entering  into  the  struc- 
ture of  plants  and  animals  it  follows  that  the  discussions  of 
physiological  chemistry  are,  in  the  main,  but  special  cases 
of  the  general  field  of  organic  chemistry.  The  important 
nutrients,  the  carbohydrates,  the  fats  and  the  protein  sub- 
stances, are  all  organic  and  the  products  appearing  as  stages 
in  their  metabolism  are  also  organic.  Therefore  much  which 
the  student  has  met  with  in  his  study  of  organic  chemistry 
may  be  found  repeated  in  his  work  in  physiological  chemistry.- 
Inasmuch  as  many  of  the  relations  to  be  now  traced  out  are 
quantitative,  it  is  highly  important,  also,  that  the  student 
should  bring  to  the  work  before  him  a  good  knowledge  of  the 
principles  of  volumetric  analysis,  as  these  will  be  applied  fre- 
quently in  what  is  to  follow. 

Historical.  To  trace  the  beginnings  of  Physiological 
Chemistry  we  are  not  obliged  to  go  far  back  in  the  develop- 
ment of  science.  \\'ith  the  old  medical  chemistry  of  the 
so-called  iatro  school  it  has  nothing  in  common,  and  in  fact 
is  in  no  sense  a  development  of  that  science  of  the  sixteenth 
and  seventeenth  centuries.  Before  the  days  of  Lavoisier,  it  is 
true,  some  little  advance  had  been  made  in  the  study  of  bodies 
of  animal  or  vegetable  origin,  but  without  a  rational  theory 
of  chemical  combination  the  isolated  facts  established  led  to 
little  of  real  value.     \\'ith  the  nature  of  respiration  explained, 


INTRODUCTION.  ■  5 

however,  and  the  identification  of  its  phenomena  with  other 
phenomena  of  oxidation,  the  way  was  opened  for  true  sci- 
entific progress.  At  the  same  time  accurate  methods  of  uki- 
mate  organic  analysis  were  suggested  and  soon  developed  by 
the  followers  of  Lavoisier.  In  the  hands  of  Berzelius,  Gmelin 
and  others  these  soon  began  to  furnish  results.  This  brings 
us  to  the  end  of  the  first  quarter  of  the  nineteenth  century,  a 
point  which  marks  the  real  beginning  of  our  science.  A  pecu- 
liar distinction  between  inorganic  and  organic  bodies  had 
gradually  arisen,  and  had  come  to  be  commonly  accepted. 
This  was  founded  on  the  notion  that  while  the  former  might 
be  produced  by  laboratory  processes  synthetically,  for  the 
latter  group  nothing  similar  was  possible.  By  this  arbitrary 
limitation  research  was  naturally  greatly  curtailed.  However, 
in  1828,  Wohler  made  the  important  discovery  that  urea  could 
be  easily  formed  by  warming  a  solution  of  ammonium  cyanate, 
and  this  was  in  time  followed  by  others  of  equal  value,  point- 
ing to  the  same  conclusion,  that  the  production  of  organic 
compounds  is  in  no  wise  dependent  on  the  aid  of  a  so-called 
vital  force.  It  was  soon  demonstrated  that  the  chemist's  labo- 
ratory, no  less  than  nature's  laboratory,  could  take  part  in  the 
formation  of  these  substances,  and  in  the  next  ten  years,  to 
about  1840,  many  products  of  physiological  interest  were 
made.  The  great  Wohler  made  many  of  the  most  fruitful 
discoveries  of  this  epoch,  but  it  is  to  Liebig  that  we  owe  the 
most.  For  many  years  he  was  busily  engaged  in  perfecting 
methods  of  analysis,  and  with  these  developed  he  turned  his 
attention  largely  to  the  chemical  phenomena  of  vegetable 
and  animal  life.  This  led  to  the  publication,  in  1840,  of  his 
epoch-marking  work,  "  Organic  Chemistry  in  Its  Relations  to 
Agriculture  and  Physiology."  This  was  followed  in  1842 
by  a  work  giving  evidence  of  his  broadened  and  strengthened 
views,  "  Organic  Chemistry  in  Its  Relations  to  Physiology 
and  Pathology."  These  works  passed  through  many  editions 
and  were  translated  into  several  languages.  In  them  we  find 
much  that  is  now  considered  fundamental  in  physiology  and 


6  PHYSIOLOGICAL    CHEMISTRY. 

physiological  chemistry,  and  they  suggested  or  called  out  the 
active  efforts  of  many  succeeding  investigators.  Not  a  few  of 
these  men  are  still  living,  so  young  is  our  science,  and  it  will 
be  sufficient  to  merely  call  attention  to  the  names  of  the  more 
prominent  workers  who  followed  the  active  pioneers.  In 
Germany  C.  G.  Lehmann  made  many  important  contributions 
to  the  chemistry  of  the  blood  and  published  a  text-book  of 
Physiological  Chemistry  which  reached  a  third  edition  in  1853. 
In  1858  F.  Hoppe-Seyler  published  the  first  edition  of  his 
Physiological  and  Pathological  Chemical  Analysis,  and  from 
1877-81  his  Physiological  Chemistry  in  four  parts  or  volumes. 
This  work  contributed  greatly  to  our  systematic  knowledge. 
C.  Voit,  since  1863  professor  of  physiology  in  Munich,  began 
his  valuable  studies  in  nutrition  and  metabolism  about  1856 
and  continued  them  nearly  forty  years.  W.  Kiihne,  of  Heidel- 
berg, did  much  to  develop  the  chemistry  of  the  protein  sub- 
stances, his  studies  dating  from  1859.  The  pupils  of  these 
German  scholars  are  to-day  among  the  most  active  investi- 
gators in  all  fields  of  physiological  chemistry. 

In  France,  Pasteur  must  be  mentioned  in  this  connection 
on  account  of  his  pioneer  investigations  on  fermentation  and 
ferments,  a  subject  of  far-reaching  importance.  CI.  Bernard 
investigated  the  chemistry  of  the  digestive  secretions  and  espe- 
cially the  behavior  of  sugar  in  the  organism.  He  published 
valuable  works  in  1853  and  1855,  which  went  through  later 
editions.  Somewhat  later  P.  Schuetzenberger,  in  Paris,  made 
important  additions  to  our  knowledge  of  the  chemistry  of  the 
protein  bodies  and  published  a  work  on  fermentation  which 
for  many  years  ranked  as  our  only  systematic  treatise  on  the 
subject. 

At  the  present  time  physiological  chemistry  has  become  a 
recognized  department  of  study  in  the  United  States,  England 
and  other  continental  countries  as  well  as  in  Germany  and 
France,  and  journals  are  now  published  devoted  solely  to  its 
interests.  The  rapidly  increasing  number  of  investigations 
published  in  these  journals  and  elsewhere  attest  the  growing 


INTRODUCTION.  7 

importance  of  the  science  from  the  theoretical  standpoint  as 
well  as  in  its  practical  relations  to  medicine. 

It  remains  to  briefly  mention  the  development  of  another 
field  of  scientific  study  because  of  its  bearing  on  certain  prob- 
lems of  physiological  chemistry.  In  the  last  quarter  of  the 
eighteenth  century  Lavoisier  clearly  showed  the  nature  of 
combustion  and  the  relation  of  animal  heat  to  respiration  and 
the  oxidation  of  the  tissues.  Lavoisier  and  Laplace  carried 
out  the  first  quantitative  experiments  in  which  a  calorimeter 
was  employed  to  measure  the  evolution  of  body  heat.  These 
were  repeated  by  Despretz  in  1824  and  later  by  Dulong. 
Since  then  by  greatly  improved  methods  many  similar  inves- 
tigations have  been  made. 

A  little  later  than  the  date  on  which  Lavoisier  and  Laplace 
announced  their  important  researches  on  the  relation  of  animal 
heat  to  oxidation  of  foodstuffs,  Benjamin  Thompson,  Count 
Rumford,  announced  a  discovery  of  equally  far-reaching  con- 
sequences. He  made  the  observation  that  the  heat  of  friction 
between  two  pieces  of  metal  may  be  absorbed  by  water  and 
so  measured,  and  that  there  is  a  relation  between  the  mechan- 
ical work  lost  in  the  friction  and  the  heat  generated.  He 
made  also  the  curious  observation  that  the  work  performed  by 
the  horse  in  one  of  his  experiments  in  which  friction  was  pro- 
duced depended  in  turn  on  the  combustion  or  oxidation  of  the 
food  of  the  horse,  from  which  it  followed  that  indirectly  the 
heating  of  the  water  was  due  to  the  combustion  of  a  certain 
amount  of  food.  But  all  the  consequences  of  his  experiments 
Rumford  did  not  see.  He  was  mainly  interested  in  showing 
the  absurdity  of  the  notion  of  the  material  nature  of  heat,  then 
commonly  held,  which  he  did  completely.  It  remained  for 
Joule  of  England  and  Mayer  of  Germany  to  point  out,  nearly 
fifty  years  later,  the  true  relation  between  heat  and  work.  In 
fine  by  establishing  the  work  equivalent  of  heat  they  made  it 
possible  to  calculate  the  food  equivalent  of  work,  since  the 
food  equivalent  of  heat  had  been  already  proven.  These  rela- 
tions are  all  of  the  highest  value  in  the  study  of  metabolism, 
to  be  considered  in  the  sequel. 


8  PHYSIOLOGICAL    CHEMISTRY. 

The  discussions  of  the  earHer  part  of  the  eighteenth  century 
placed  in  clear  light  finally  the  full  meaning  of  the  doctrine 
of  the  Indestructibility  of  Matter.  These  later  discussions 
developed  a  new  doctrine,  that  of  the  Conservation  of  Energy, 
the  recognition  of  which  played  no  small  part  in  the  gradual 
advance  of  physiological  as  well  as  physical  science. 

It  is  the  intention  of  the  following  chapters  to  present  the 
fundamental  facts  and  theories  of  physiological  chemistry  in 
the  simplest  possible  manner.  ^luch  matter  found  in  the 
larger  hand-books  must  necessarily,  therefore,  be  omitted  from 
a  work  of  this  elementary  character.  But  enough  will  be 
given  to  furnish  the  student,  it  is  hoped,  a  satisfactory  view  of 
that  which  is  most  important  in  the  science  at  the  present  time. 
It  will  be  found  convenient  to  make  four  general  divisions  of 
the  subject,  as  follows : 

Section  I.     The  Nutritives  and  Related  Substances. 

Section  II.     Ferments  and  Digestive  Processes. 

Section  III.  The  Chemistry  of  the  Tissues  and  Secre- 
tions of  the  Body. 

Section  IV.  The  End  Products  of  Metabolism.  Ex- 
cretions.    Energy  Balance. 


SECTION    I. 


CHAPTER    II. 
THE  NUTRITIVES. 

INORGANIC  ELEMENTS.  WATER.  AIR.  SALTS. 

Composition  of  the  Body.  The  Hving-  animal  body  is 
composed  in  the  mean  of  about  35  to  40  per  cent  of  sohds 
and  60  to  65  per  cent  of  water.  In  adults  the  solids  are 
somewhat  in  excess  of  this  amount,  while  in  infants  they  are 
lower,  perhaps  not  over  30  per  cent.  The  elements  most 
abundantly  present  are  carbon,  oxygen,  hydrogen,  nitrogen, 
phosphorus,  sulphur,  chlorine,  potassium,  sodium,  calcium, 
magnesium  and  iron.  In  traces  only,  or  in  particular  tissues, 
we  find  iodine,  fluorine,  bromine,  silicon,  manganese,  copper 
and  lithium.  The  presence  of  these  in  minute  amount  seems 
to  be  necessary  for  the  existence  of  certain  animals.  These 
elements  are  not  present  in  the  free  state,  but  exist  combined 
in  more  or  less  complex  compounds,  the  degree  of  complexity 
varying  between  that  illustrated  in  such  simple  bodies  as  water 
or  common  salt,  and  that  found  in  the  large  protein  molecules 
with  possibly  thousands  of  atoms  present. 

In  point  of  abundance  these  elements  are  found  in  the  body 
in  about  the  following  order : 

Table  of  the  Elements  in  the  Body. 


Name  of  Element. 

Per  Cent. 
Amount. 

Occurrence. 

Oxygen 
Carbon 
Hydrogen 

66.0 

I7-S 
10.2 

In  the  water  of  the  body,  in  the  fats,  the  pro- 
tein substances  and  in  nearly  all  the  tissues 

and  salts. 
In  the  fats,  protein  substances  and  in  most  of 

the    important   compounds   produced    in   the 

body. 
In  water,  the   fats,  protein   substances   and  in 

the  important  products  of  metabolism. 

lO 


PHYSIOLOGICAL    CHEMISTRY, 


Name  of  Element. 

Per  cent 
Amount. 

Nitrogen 

2.4 

Calcium 

1.6 

Phosphorus 

0.9 

Potassium 

0.4 

Sodium 

0.3 

Chlorine 

0.3 

Sulphur 

0.2 

Magnesium 

0.05 

Iron 

0.004 

Iodine,  Fluorine, 
Silicon 

>  traces. 

Occurrence. 


Found  mainly  in  the  protein  substances  of  the 
body.  Also  in  many  of  the  metabolic  prod- 
ucts derived  from  these. 

This  element  occurs  mainly  in  the  bones,  but 
is  found  in  the  blood  also  and  in  several 
secretions  in  small  amount. 

Is  found  principally  with  calcium  in  the  bones, 
but  occurs  also  in  several  complex  com- 
pounds in  organic  combination. 

Found  as  chloride,  carbonate  or  phosphate  in 
many  of  the  body  tissues  and  secretions. 
Exists  also  in  organic  combination. 

Occurs  combined,  as  does  potassium;  the 
chloride  and  carbonate  are  the  most  im- 
portant salts  and  are  found  in  several  body 
fluids. 

Foimd  in  combination  with  sodium  and  potas- 
sium, also  as  hydrochloric  acid  in  the  gastric 
juice. 

This  element  is  important  as  occurring  in  the 
protein  compounds  of  the  body,  and  is  found 
also  in  minute  amount  in  other  combinations. 

Is  found  mainly  as  phosphate  and  carbonate  in 
the  bones. 

Iron  occurs  in  the  important  hemoglobin  of 
the  lilood,  as  an  integral  part  of  the  complex 
molecule.  It  is  found  also  in  inorganic  com- 
pounds in  traces. 

Fluorine  is  found  in  the  teeth,  iodine  in  the 
thyroid  gland,  silicon  in  the  hair.  Besides 
these,  other  elements  have  been  found  occa- 
sionally, but  do  not  appear  to  be  necessary. 


The  solids  of  the  body  are  therefore  both  organic  and  inor- 
ganic, and  approximately  the  composition  of  the  whole  may 
be  thus  represented : 

Per  Cent. 

Water 65 

Protein  substances IS 

Fats  14 

Other  organic  extractives i 

Mineral   matters 5 


It  must  be  remembered,  however,  that  the  fat  may  vary 
widely  from  the  above  number  and  therefore  change  the  ratio, 
fat:  protein.  i\mong  the  mineral  matters  calcium  phosphate 
holds  the  first  place,  as  it  makes  up  the  larger  part  of  bone 


INORGANIC    ELEMENTS.  I  I 

ash ;  carbonates  and  chlorides  of  the  alkah  metals  make  up  the 
remainder  largely. 

In  view  of  this  composition  of  the  body,  it  is  important  to 
learn  how  its  waste  is  replenished,  and  what  substances  must 
be  or  may  be  consumed  to  repair  the  constant  losses  and 
enable  the  body  to  do  its  proper  work.  This  leads  to  the 
question  of  foods  or  nutritives  in  the  broad  sense.  Beginning 
with  the  inorganic  materials  used  by  the  body,  we  have  first : 

Water.  As  it  appears  on  the  surface  of  the  earth  water 
is  classed  conveniently  as  hard  and  soft.  The  descending 
rain,  after  the  dust  is  washed  from  the  air,  consists  of  nearly 
chemically  pure  water.  It  holds  no  mineral  matters  dissolved, 
and  is  contaminated  mainly  with  a  small  amount  of  dissolved 
carbon  dioxide.  Such  water  on  reaching  the  earth  is  soft  and 
can  replace  distilled  water  for  most  purposes.  The  changes 
which  follow  after  contact  with  the  soil  depend  on  the  compo- 
sition of  the  latter.  If  the  strata  over  which  the  water  flows 
or  through  which  it  percolates  consist  of  sand,  quartz  or 
silicate  rocks  or  other  insoluble  materials  the  water  is  left 
in  practically  pure  condition,  and  is  the  water  usually  spoken 
of  as  soft  water.  But,  on  the  other  hand,  if  the  rain  water 
comes  in  contact  with  limestone,  gypsum,  or  other  slightly 
soluble  substances,  something  goes  into  solution  and  the 
product  is  now  known  as  hard  water,  the  degree  of  "  hard- 
ness "  depending  on  the  amount  of  dissolved  solids.  Waters 
containing  the  carbonates  of  calcium  and  magnesium  are  de- 
scribed as  temporarily  hard,  since  the  carbonic  acid  which 
holds  these  carbonates  in  solution  may  be  removed  by  boiling, 
which  causes  precipitation.  Calcium  sulphate  or  chloride  in 
water  can  not  be  precipitated  by  boiling  and  the  presence  of 
these  and  a  few  other  substances  produces  permanent  hardness. 

Moderate  amounts  of  these  mineral  matters  in  water  are  not 
objectionable;  in  fact  waters  with  some  lime  and  magnesia 
are  preferable  to  absolutely  soft  water  for  drinking  purposes. 
But  along  with  the  inorganic  substances  the  water  may  take 
other  things  from  the  soils  with  which  it  comes  in  contact  that 


12  PHYSIOLOGICAL    CHEMISTRY. 

are  not  so  desirable.  These  are  various  partly  decomposed 
organic  matters  of  animal  or  vegetable  origin  and.  what  is 
more  important,  minute  living  vegetable  cells,  mostly  bacteria, 
which  are  capable  of  causing  much  mischief  when  taken  into 
the  stomach  of  man.  It  is  very  generally  believed  that  sev- 
eral diseases  in  man  have  their  origin  in  the  consumption  of 
water  contaminated  in  this  way. 

Natural  Purification  of  Water.  But  it  must  not  be  sup- 
posed that  these  bacteria  are  always  harmful.  On  the  con- 
trary some  of  them  are  the  common  agents  which  effect  the 
natural  purification  of  waters  containing  organic  matter,  in 
which  they  incite  destructive  fermentation  or  putrefactive 
changes  and  finally  oxidation.  As  a  result  of  these  changes 
harmless  inert  substances  such  as  nitrogen  or  nitrates,  carbon 
dioxide,  methane  and  water  are  produced  from  the  relatively 
complex  waste  or  excreta  of  the  higher  organisms.  \\'hen 
we  speak  of  the  spontaneous  or  self-purification  of  water  we 
refer  to  a  series  of  changes  in  which  these  bacteria  play  a 
leading  part. 

Artificial  Purification  of  Water.  On  a  smaller  scale 
water  may  be  rendered  safe  and  suitable  for  household  use  by 
several  methods.  By  distillatioji  all  objectionable  matters 
may  be  rejected  and  a  wholesome  drinking  water  obtained.  It 
is  possible,  also,  to  separate  practically  all  bacteria  and  other 
solid  matters  by  filtration  through  beds  of  fine  sand.  In  this 
way  the  supplies  of  many  cities  are  obtained  at  the  present 
time.  Frequently  the  filtration  is  preceded  by  coagulation  or 
precipitation  of  the  organic  substances  by  means  of  some 
suitable  agent  such  as  alum  or  salts  of  iron,  or  lime. 

Tests  of  Drinking  Water.  In  the  sanitary  examination 
of  water  it  is  not  necessary  to  make  very  full  analyses  to  deter- 
mine its  value  for  household  use.  A  few  tests  usually  suffice 
to  discover  the  presence  or  absence  of  objectionable  substances. 
For  example,  in  uncontaminated  waters  from  ordinary  springs, 
lakes,  rivers  or  wells,  chlorine  is  present  in  small  amount  only. 
Any  excess  of  chloride  suggests  contact  with  sewage  or  house- 


INORGANIC    ELEMENTS.  13 

hold  waste  somewhere,  and  a  quantitative  test  is  of  prime 
importance  to  settle  this  point.  Such  a  test  and  a  few  others 
will  be  illustrated  below. 

Ex.  The  Test  for  Chlorides.  A  test  is  often  made  in  this  way: 
Measure  out  200  cc.  of  the  water,  add  to  it  a  few  drops  of  a  solution  of 
pure  neutral  potassium  chromate,  and  then  from  a  burette  run  in,  with 
constant  stirring,  solution  of  tenth  normal  silver  nitrate  until  a  faint  red- 
dish precipitate  of  silver  chromate  appears.  Each  cubic  centimeter  of  the 
silver  solution  precipitates  3.54  mg.  of  chlorine  from  common  salt  or  other 
chloride,  and  when  the  last  trace  of  chlorine  is  combined,  the  silver  begins 
to  precipitate  the  chromate  with  production  of  red  color.  The  chromate 
acts  here  as  an  "indicator,"  as  it  shows  just  when  the  chlorine  is  all  com- 
bined by  beginning  to  precipitate  itself. 

In  making  this  test  it  is  well  to  take  two  similar  beakers,  place  them 
side  by  side  on  white  paper,  pour  equal  amounts  of  water  in  each,  add  to 
each  the  same  number  of  drops  of  the  indicator,  and  then  with  one  make 
the  actual  test  by  adding  the  silver  solution.  Note  the  amount  used  to 
give  a  light  shade  and  then  discharge  it  by  adding  a  drop  of  salt  solution. 
Now,  with  this  opalescent  or  turbid  liquid  for  comparison  add  silver  nitrate 
to  the  second  beaker  tmtil  the  light  yellowish  red  shade  just  appears.  This 
reading  is  usually  somewhat  more  accurate  than  the  first. 

As  a  result  of  the  decomposition  of  various  nitrogenous 
matters  ammonia  is  frequently  found  in  natural  waters.  Its 
amount  is  therefore  a  measure  of  contamination  to  some  ex- 
tent, and  tests  for  its  presence  are  always  made  in  sanitary 
examinations.  In  practice  the  test  is  usually  made  on  a  dis- 
tillate from  the  water  in  question,  but  the  following  experi- 
ment will  illustrate  the  behavior  of  the  reagent  employed. 

Ex.  Test  for  Ammonia.  Solutions  of  ammonia  or  ammonium  salts 
possess  the  peculiar  property  of  giving  a  yellowish  brown  color  with  what 
is  known  as  Nessler's  reagent  (a  solution  of  mercuric-potassium  iodide, 
made  strongly  alkaline  with  sodium  or  potassium  hydroxide).  With  more 
than  traces  of  ammonia  a  precipitate  is  formed. 

To  make  the  test  measure  out  50  cc.  of  the  water  in  a  large  test-tube, 
or  tall  narrow  beaker,  and  add  to  it  2  cc.  of  the  Nessler  solution.  By 
placing  the  beaker  on  a  sheet  of  white  paper  and  looking  down  through  it, 
the  depth  of  color  can  be  observed.  A  few  parts  of  ammonia  in  one  hun- 
dred million  parts  of  water  can  be  readily  seen  and  measured. 

The  Oxidaton  Tests.  Pure  water  absorbs  free  oxygeii 
from  the  atmosphere  but  has  no  tendency  to  decompose  com- 
pounds to  secure  it.     On  the  other  hand  waters  containing 


14  PHYSIOLOGICAL    CHEMISTRY. 

organic  matters  or  certain  inorganic  contaminations  have  the 
power  of  decomposing  oxygen  salts  to  secure  the  oxygen  they 
desire,  and  the  amount  of  oxygen  so  taken  up  becomes  a 
measure  of  the  impurity  of  the  water.  Potassium  perman- 
ganate is  a  salt,  which,  under  certain  conditions,  gives  up  its 
oxygen  to  waters  containing  organic  bodies  in  solution  and  is 
frequently  employed  in  water  analysis  for  this  purpose.  An 
experiment  will  show  one  way  in  which  it  is  used. 

Ex.  Measure  out  about  loo  cc.  of  pure,  carefully  distilled  water,  pour 
it  into  a  clean  beaker  in  which  water  has  just  been  boiled  and  add  5  cc. 
of  pure  dilute  sulphuric  acid  (i  to  3).  Place  the  beaker  on  wire  gauze 
and  heat  to  boiling.  Now  add  5  drops  of  a  dilute  permanganate  solution 
(300  milligrams  to  the  liter)  from  a  burette  or  dropping  tube  and  boil  five 
minutes.     The  pink  color  persists. 

Repeat  the  experiment,  using  100  cc.  of  common  hydrant  water  to  which 
a  trace  of  egg  albumin  or  urea  has  been  added,  and  after  running  in  the 
permanganate  boil  again.  The  color  fades  out  and  more  may  be  added. 
Finally,  after  sufficient  has  been  added  the  pink  color  remains.  The  num- 
ber of  drops  or  cubic  centimeters  used  is  a  measure  of  the  contamination 
of  the  water,  although  often,  as  in  this  experiment,  a  very  rough  one. 

The  Tests  for  Nitrites  and  Nitrates.  Nitrogenous 
matters  undergoing  oxidation  in  water  and  soil  usually  give 
rise,  in  time,  to  nitrites  and  finally  to  nitrates.  These  com- 
pounds are  therefore  looked  for  in  water  as  evidence  of  past 
contamination.  In  most  instances  nitrites,  as  a  less  advanced 
stage  of  oxidation  than  nitrates,  suggest  comparatively  recent 
contamination.  The  tests  are  especially  interesting  in  the 
examination  of  well  and  spring  water. 

Chemists  are  acquainted  with  a  number  of  methods  for  the 
detection  of  traces  of  nitrogen  in  the  form  of  nitrites  and 
nitrates,  but  at  the  present  time  certain  color  reactions  are, 
because  of  their  simplicity,  mainly  in  favor.  These  are  illus- 
trated by  the  following  tests : 

A  reagent  for  nitrites  is  prepared  by  dissolving  0.5  gm.  of 
sulphanilic  acid  in  150  cc.  of  acetic  acid  of  25  per  cent  strength, 
and  mixing  this  with  a  solution  of  o.i  gm.  of  pure  naphthyla- 
mine  in  200  cc.  of  dilute  acetic  acid.  This  mixture  keeps  very 
well  for  a  time  in  the  dark. 


INORGANIC    ELEMENTS.  I  5 

Ex.  To  about  50  cc.  of  water  in  a  clean  beaker  add  2  cc.  of  the  above 
solution.  If  the  water  is  quite  free  from  nitrites  the  reagent  imparts  no 
color  to  it.  One  hundredth  of  a  milligram  of  nitrogen  as  nitrite  in  the 
water  gives  a  faint  pink  color  at  the  end  of  five  minutes ;  with  large  quan- 
tities the  color  may  become  deep  rose  red. 

Ex.  A  nitrate  test  may  be  illustrated  in  this  manner :  To  the  residue 
obtained  by  evaporating  50  cc.  of  an  ordinary  river  or  lake  water  to  dry- 
ness in  a  porcelain  dish  add  i  cc.  of  phenolsulphonic  acid.  Rub  the  acid 
over  the  bottom  of  the  dish,  and  add  a  few  drops  of  dilute  sulphuric  acid. 
Warm  the  dish  a  few  minutes  and  add  25  cc.  of  water.  This  should  show 
now  a  faint  yellow  color.  By  supersaturating  with  ammonia  the  color  be- 
comes deeper.  In  this  experiment  picric  acid  is  at  first  formed  if  a  nitrate 
is  present  and  the  addition  of  ammonia  yields  ammonium  picrate,  the  color 
of  which  is  more  marked. 

For  the  interpretation  of  all  these  tests  works  on  sanitary  analysis  must 
be  consulted. 

Physiological  Importance  o£  Water.  This  is  suggested 
by  the  large  proportion  in  which  it  is  present  in  the  animal 
body,  as  shown  above.  It  senses  primarily  as  the  general 
solvent  for  all  the  solid  foodstuffs  taken  into  the  system  and 
assists  in  the  removal  of  the  solid  waste  products  or  excreta. 
To  accomplish  these  ends  it  must  be  drunk  in  sufficient  quan- 
tity. It  is  a  well  recognized  fact  that  most  people  in  the  United 
States  drink  too  little  water,  from  which  various  ills  result. 
Important  chemical  changes  within  the  body  are  dependent  on 
the  so-called  hydrolytic  action  of  water.  These  appear  mainly 
in  the  phenomena  of  digestion,  in  which  starches,  sugars,  pro- 
tein bodies  and  fats  are  altered  before  absorption,  and  will  be 
discussed  in  detail  in  sections  to  follow. 

It  must  be  remembered  further  that  water  plays  a  very 
important  part  in  the  removal  of  heat  from  the  body.  For 
each  gram  of  water  evaporated  as  perspiration  or  in  the 
breath  nearly  600  units  of  heat  are  absorbed  and  in  this  way 
over  20  per  cent  of  the  heat  expenditure  may  be  accounted  for. 

The  average  amounts  of  water  found  in  the  important  tissues 
is  shown  in  the  following  table: 

Per  Cent.  Per  Cent. 

Dentine    10  Elastic  tissue    50 

Fatty  tissues    20  Liver   70 

Bones    50  Skin  72 


1 6  PHYSIOLOGICAL    CHEMISTRY. 

Per  Cent.  Per  Cent. 

Muscles    75  Brain    (gray  matter)..  86 

Spleen 76  Milk   88 

Pancreas    78  Vitreous  humor   98.5 

Blood    79  Cerebro-spinal   fluid    . .  99.0 

Kidney  83  Saliva    99.5 

Air,  Besides  its  content  of  oxygen,  nitrogen  and  argon 
the  atmosphere  contains  several  other  gases  in  small  amount. 
The  most  abundant  of  these  is  water  vapor,  with  smaller  traces 
of  carbon  dioxide,  helium,  neon,  etc.  As  the  amount  of 
aqueous  vapor  present  is  extremely  variable  it  is  customary  to 
give  the  analysis  of  the  dry  air  only,  which  in  volume  per 
cent  is  about  this,  the  rarer  gases  being  included  with  the 
nitrogen  and  the  argon  : 

Per  Cent. 

Nitrogen    78.40 

Oxj'gen    20.94 

Argon    0.63 

Carbon  dioxide    03 

The  water  vapor  present  varies  with  the  temperature  and 
other  physical  conditions  and  may  sometimes  make  up  one 
per  cent,  or  even  more,  by  weight  of  the  whole  mass.  A 
cubic  meter  of  fully  saturated  air  contains  30.1  grams  of 
aqueous  vapor  at  30 '^  C,  and  75  per  cent  of  this  is  frequently 
present  in  the  hot,  "  close  "  weather  of  our  summers.  It  is 
this  high  proportion  of  moisture  which  renders  further  evap- 
oration from  the  skin  so  difficult,  and  which  therefore  contrib- 
utes greatly  to  our  bodily  discomfort. 

The  normal  carbon  dioxide  content  is  given  above  as  0.03 
per  cent  or  three  cubic  centimeters  in  ten  liters.  This  amount 
is  greatly  exceeded  in  the  air  of  poorly  ventilated  houses,  but 
is  not  in  itself  the  cause  of  the  unpleasant  sensations  expe- 
rienced in  going  into  such  an  atmosphere,  although  this  was 
long  believed.  It  has  been  found  by  experiment  that  one  can 
breathe,  although  not  comfortably,  in  a  pure  atmosphere  con- 
taining as  much  as  3  per  cent  of  carbon  dioxide,  while  an 
atmosphere  contaminated  to  the  extent  of  i  per  cent  by  human 


INORGANIC    ELEMENTS.  I  7 

respiration  would  be  practically  unbearable.  This  condition 
is  doubtless  due  to  the  traces  of  organic  products  throAvn 
off  in  the  breath  and  perspiration,  and  especially  to  the  decom- 
position of  organic  matter  on  the  unclean  skin.  The  carbon 
dioxide  is  often  made  the  approximate  measure  of  the  con- 
tamination of  inhabited  rooms,  because  of  the  practical  diffi- 
culty of  measuring  anything  else. 

In  respiration  the  air  is  modified  about  as  shown  by  these 
figures : 

Inspired  Air         Expired  Air 
Per  Cent.  Per  Cent. 

Nitrogen,    argon,    etc 79.0  80.0 

Oxj'gen    21.0  16.0 

Carbon    dioxide    03  4.0 

The  amount  of  oxygen  inhaled  each  day  by  a  full  grown 
man  is  not  far  from  500  liters,  while  the  volume  of  carbon 
dioxide  exhaled  is  somewhat  less,  about  450  liters  in  the 
mean.  Later  something  will  be  said  about  the  numerical  rela- 
tion existing  between  the  volume  of  carbon  dioxide  eliminated 
and  the  volume  of  oxygen  absorbed. 

The  most  accurate  method  of  finding  the  amounts  of 
aqueous  vapor  and  carbon  dioxide  in  the  air  is  to  aspirate  a 
measured  volume  through  a  series  of  weighed  absorption 
tubes.  The  first  of  these  contain  dry  granular  calcium 
chloride  or  some  other  good  water  absorbent,  while  the  fol- 
lowing tubes  contain  soda-lime  or  a  strong  potassium  hydrox- 
ide solution  to  absorb  the  carbon  dioxide.  The  increase  in 
weight  of  the  tubes  shows  the  amount  of  vapor  and  gas 
absorbed  from  the  given  volume  of  air.  For  quick  determi- 
nations somewhat  less  exact  methods  are  often  used  in  practice. 

The  atmosphere  often  contains  traces  of  other  gases,  as 
ammonia,  sulphurous  oxide,  oxides  of  nitrogen  and  ozone, 
which  are  of  little  physiological  importance  and  need  not  be 
here  considered.  Of  greater  importance  are  the  minute 
organised  forms  everywhere  present  to  some  extent,  at  least, 
and  which  include  bacteria  and  many  other  agents  of  putre- 
faction and  fermentation.  Most  of  these  are  practically  harm- 
3 


1 8  PHYSIOLOGICAL    CHEMISTRY. 

less  in  respiration,  but  the  presence  of  others  is  an  element  of 
the  greatest  danger,  because  of  the  disturbances  they  occasion 
when  taken  into  the  body. 

MINERAL    SUBSTANCES    REQUIRED. 

Salts.  The  table  some  pages  back  gives  the  percentage 
amount  of  the  dififerent  elements  which  make  up  the  human 
body,  some  being  united  in  organic  and  the  others  in  inorganic 
compounds.  Aside  from  water  the  most  abundant  and  im- 
portant of  the  inorganic  materials  are  the  phosphates  and 
carbonates  of  the  alkali-earth  metals  found  in  the  bones,  the 
alkali  chlorides  and  the  alkali  carbonates.  The  solid  mineral 
matter  or  ash  of  the  adult  body  amounts  in  the  mean  to  about 
5  per  cent ;  not  far  from  four-fifths  of  this  content  comes 
from  the  skeleton,  while  about  one-tenth  of  it  is  derived  from 
the  muscles.  The  proportion  of  ash  in  the  different  tissues, 
taken  in  the  moist  condition,  is  approximately  as  follows : 

Per  Cent.  Per  Cent. 

Bones   33  Pancreas,  brain i.o 

Cartilage   2  Lung,  heart   0.95 

Liver  and  spleen 1.5  Blood  0.93 

Muscles    1.3  Skin    075 

Kidney  1.2  Milk   0.70 

Leaving  traces  out  of  consideration,  it  appears  that  the  body 
contains  four  metallic  elements,  calcium,  sodium,  potassium 
and  magnesium,  which  exist  in  combination  with  four  acids, 
viz.,  phosphoric,  hydrochloric,  carbonic  and  sulphuric.  Of 
all  these  compounds  the  calcium  phosphate  of  the  bones  is 
the  most  abundant. 

Phosphates.  The  phosphates  of  the  body  are  salts  of  the 
common  or  orthophosphoric  acid,  H3PO4.  The  three  kinds 
of  salts  possible  here  are : 

Primary  phosphates,  ]MH2P04, 
Secondary  phosphates,  M2HPO4, 
Tertiary  phosphates,       AlaPOi. 


INORGANIC    ELEMENTS.  1 9 

The  alkali  salts  of  the  three  classes  are  readily  soluble  in 
water.  The  secondary  and  tertiary  phosphates  are  mostly  in- 
soluble, those  of  the  alkali  metals  excepted.  Secondary  phos- 
phates are  converted  into  pyrophosphates  by  heat  and  the 
primary  phosphates  into  metaphosphates.  To  most  indica- 
tors the  primary  phosphates  shoAV  acid  behavior,  while  the  sec- 
ondary phosphates  are  feebly  alkaline.  The  soluble  tertiary 
phosphates  are  strongly  alkaline.  The  action  on  different 
indicators  must  be  remembered  in  attempting  to  estimate  the 
acidity  or  alkalinity  of  urine. 

The  phosphates  furnished  us  in  various  animal  and  vege- 
table foods  are  mainly  those  of  calcium  and  potassium,  but  it 
is  likely  that  the  larger  part  of  the  phosphorus  utilized  in  the 
body  is  combined  in  relatively  complex  organic  compounds, 
the  lecithins  and  nucleins,  for  example,  which  yield  phosphoric 
acid  and  phosphate  in  the  final  oxidation.  AVe  find  therefore 
the  tertiary  calcium  and  magnesium  phosphates,  Ca3(P04)2 
and  Mg3(P04)2  in  bones.  Acid  calcium  phosphate  of  the 
formula  Ca(H2P04)2  occurs  in  some  of  the  body  fluids,  and 
is  an  important  urinary  excretion.  The  phosphate  CaHP04 
may  sometimes  be  deposited  from  the  urine.  Secondary  po- 
tassium phosphate,  K2HPO4,  is  a  constituent  of  all  animal 
cell  structures,  possibly  in  soluble  form,  but  possibly,  also,  in 
organic  combination.  The  muscular  juice  is  rich  in  alkali 
phosphates. 

Chlorides.  Chlorine  is  found  in  the  body  as  sodium 
chloride  and  potassium  chloride,  also  as  free  hydrochloric  acid 
in  the  gastric  juice.  In  our  foodstuffs  it  comes  to  us  mainly 
as  sodium  chloride,  but  this  by  double  decomposition  may 
give  rise  to  the  potassium  chloride  later : 

K.CO3  +  2NaCl  =  2KCI  -f  NaoCOs. 

In  the  gastric  and  pancreatic  juices  and  in  the  blood  sodium 
chloride  is  more  abundant  than  potassium  chloride,  but  the 
latter  is  in  excess  in  the  cell  structures.  Chlorine  is  utilized 
in  the  animal  body  only  as  it  is  found  in  the  metallic  com- 


20  PHYSIOLOGICAL    CHEMISTRY. 

pounds  or  chlorides.  The  various  organic  combinations  of 
chlorine  can  not  replace  the  salts.  It  has  been  pointed  out 
by  Bunge  that  sodium  chloride  is  much  more  necessary  in  the 
food  of  man  or  animals  consuming  a  vegetable  diet  than  it  is 
when  the  diet  is  mainly  flesh. 

Carbonates,  The  carbonates  found  in  the  human  body  are 
produced  there  from  the  carbonic  acid  of  oxidation.  Hard 
waters  contain  the  carbonates  of  calcium  and  magnesium,  but 
these  must  suffer  decomposition  when  taken  into  the  stomach, 
and  the  traces  of  acid  gas  so  expelled.  Besides  the  carbon 
dioxide  of  tissue  oxidation  we  must  consider  also  that  formed 
by  several  ferment  processes  in  the  intestines.  A  large 
amount  of  the  gas  is  produced  in  this  way  and  part  of  this 
is  absorbed  into  the  circulation.  Under  certain  conditions  the 
"  weak  "  carbonic  acid  is  able  to  decompose  sodium  chloride 
and  produce  sodium  carbonate  and  free  hydrochloric  acid. 
The  origin  of  the  latter  in  the  gastric  juice  is  now  accounted 
for  in  this  way.  while  the  sodium  carbonate  formed  at  the 
same  time  is  carried  into  the  blood  and  through  this  to  other 
parts  of  the  body,  where  other  carbonates  may  be  made  by 
double  decompositions.  The  soluble  alkali  carbonates  are  the 
most  abundant,  but  in  the  bones  and  in  the  teeth  calcium  car- 
bonate forms  an  important  part.  Some  carbonates  are  always 
excreted  by  the  urine,  and  the  alkali-earth  carbonates  may 
occasionally  appear  in  the  sediment. 

Sulphates,  Sulphur  may  enter  the  body  in  a  variety  of 
combinations  but  nearly  all  of  it  is  finally  excreted  in  the 
completely  oxidized  form,  that  is,  as  sulphates,  by  the  urine. 
Sulphur  in  organic  combination  is  found  in  all  protein  sub- 
stances and  in  the  final  oxidation  of  these  compounds  in  the 
body  sulphuric  acid  is  produced.  The  manner  in  which  this 
is  combined  before  excretion  will  be  discussed  later.  The 
amount  of  sulphur  normally  present  in  the  body  is  but  a 
small  fraction  of  one  per  cent  of  the  weight  of  the  latter  and 
practically  all  of  it  is  found  in  the  protein  or  protein-like  sub- 
stances.    Among  these  keratin   is  characterized  bv  its  rela- 


INORGANIC    ELEMENTS.  21 

tively  high  sulphur  content.  Of  the  exact  manner  in  which 
the  sulphur  is  combined  in  most  of  these  bodies  but  little  is 
known. 

Bases.  The  several  acid  radicals  referred  to  occur  in  com- 
bination with  metals  and  four  of  these  only  are  present  in 
the  body  in  appreciable  quantity.  These  are  calcium,  mag- 
nesium, sodium  and  potassium,  and  they  exist  in  the  form  of 
salts  with  the  acid  radicals.  To  these  four  the  iron  of  the 
blood  must  be  added,  but  it  is  present  in  organic  combina- 
tion. In  a  following  chapter  it  will  be  shown  to  what  extent 
these  bases  are  present  in  ordinary  foodstuffs.  Of  some  it  is 
important  that  they  enter  the  body  in  certain  forms  only, 
otherwise  their  utilization — assimilation — is  imperfect  or  even 
impossible.  This  is  especially  true  of  the  iron,  the  chief  use 
of  which  is  in  the  building  up  of  hemoglobin.  For  this  pur- 
pose the  iron  of  the  ordinary  mineral  salts  is  not  available,  but 
it  may  be  taken  from  certain  peculiar  organic  combinations. 
Many  mineral  matters  are  as  essential  for  the  growth  of  the 
body  as  are  the  organic  foods  to  be  described  in  the  next  chap- 
ters and  care  must  be  observed  to  provide  them  in  sufficient 
quantity,  especially  in  the  feeding  of  the  young.  There  is 
some  reason  for  believing  that  most  of  the  basic  material 
assimilated  in  the  body  is  in  the  form  of  complex  salts  or 
organo-metallic  combinations  of  some  kind.  The  sulphur, 
phosphorus  and  carbon  are  so  found  and  possibly  the  metals 
also.  It  has  been  pointed  out  that  in  the  oxidation  of  such 
organo-metallic  compounds  carbonates  or  basic  salts  must 
result,  and  some  portion  at  least  of  these  salts  must  be  avail- 
able to  combine  with  the  sulphuric  acid  arising  from  the 
oxidation  of  the  protein  substances.  According  to  Bunge 
common  salt  is  the  only  mineral  substance,  in  excess  of  that 
furnished  by  the  usual  organic  foods,  which  the  body  actually 
demands  in  large  amount. 


CHAPTER    III. 

THE  CARBOHYDRATES  AND  RELATED  BODIES. 

Under  the  term  carbohydrate  it  has  long  l^een  customary  to 
inckide  a  number  of  bodies  with  closely  related  properties  and 
similar  composition,  which  may  be  expressed  by  such  smiple 
formulas  as  CcHioOj,  CcHioOc.  or  multiples  of  these.  The 
term  carbohydrate  came  into  use  long  before  the  structure  of 
the  bodies  in  question  was  known.  It  is  now  possible  to 
describe  these  substances  in  their  relations  to  the  fundamental 
hydrocarbons  or  alcohols  and  this  classification  will  be  there- 
fore briefly  explained. 

NATURE    OF    THE    CARBOHYDRATES. 

In  their  chemical  behavior  these  bodies  resemble  aldehydes 
or  ketones  in  certain  important  characteristics.  Like  the 
latter  they  are  often  strong  reducing  substances  and  most 
of  them  form  combinations  with  phenyl  hydrazine.  These 
and  other  properties  suggest  that  they  may  be  considered  as 
aldehyde  or  ketone  derivatives  of  the  polyhydric  alcohols, 
which  relationship  is  shown  by  the  following  table,  which  con- 
tains also  some  acid  derivatives  for  further  illustration. 

Some  of  the  bodies  in  the  table  are  naturally  occurring  sub- 
stances and  are  highly  important,  but  most  of  them  are  arti- 
ficial. The  aldohexoses  and  the  ketohexoses  are  closely 
related  to  two  groups  of  more  complex  bodies,  in  which  cane 
sugar  and  starch  are  the  best  illustrations,  and  with  them  form 
the  important  class  of  carbohydrates  in  the  more  restricted 
sense. 


22 


CARBOHYDRATES    AND    RELATED    BODIES. 
Relations  of  the  Carbohydrates. 


23 


Polyhydric 
Alcohols. 

Aldehyde  Deriva- 
tives. 

Ketone  Deriva- 
tives. 

Acid  Derivatives. 

Acid  Derivatives. 

CH^OH 

CHjOH 

CH^OH 

COOH 

CHjOH 
Glycol. 

CHO 
Glycol  aldehyde, 
diose. 

COOH 
Glycollic  acid. 

COOH 
Oxalic  acid. 

CHjjOH 

CHjOH 

CHjOH 

CHjOH 

COOH 

CHOH 

CHOH 

CO 

CHOH 

CHOH 

1 

CHjOH 
Glycerol. 

1 

CHO 
Glyceraldehyde. 

CHjOH 
Dioxyacetone. 

COOH 
Glyceric  acid. 

COOH 
Tartronic  acid. 

Glycerose   or  triose. 

CHjOH 

CHjOH 

CH2OH 

CH^OH 

COOH 

CHOH 

CHOH 

CHOH 

CHOH 

1 

CHOH 

CHOH 

CHOH 

CO 

CHOH 

CHOH 

CHpH 
Erythrol. 

CHO 
Aldotetrose. 

CHjOH 
Ketotetrose. 

COOH 
Erythritic  acid. 

COOH 
Tartaric  acids. 

Erythrose. 

CH^OH 

CH^OH 

CH^OH 

CH,OH 

1 

COOH 
1 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

1 

CO 

CHOH 

CHOH 

CHjOH 

Pentitols, 
arabitols,  etc. 

1 

CHO 
Aldopentoses, 
arabinose,  etc. 

CUflU 
Ketopentoses. 

COOH 
Tetrahydroxy- 
monocarboxylic 
acids,  arabonic 

acid,  etc. 

COOH 

Trihydroxydi- 

carboxylic 

acids,  trioxyglu- 

taric  acids. 

Pentoses. 

CHjOH 

CH^OH 

CH2OH 

CHjOH 

COOH 

CHOH 

1 
CHOH 

CHOH 

CHOH 

CHOH 

1 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CHOH 

CO 

CHOH 

CHOH 

CH^OH 

Hexitols, 

mannitol,etc. 

1 

CHO 
Aldohexoses,  glu- 
coses, etc. 

CHjOH 

Ketohexoses, 

fructose. 

COOH 

Pentahydroxycar- 

boxylic  acids. 

COOH 

Tetrahydroxy- 

dicarboxylic 

Hexoses. 

dextronic  acid. 

acid,  etc. 

24 


PHYSIOLOGICAL    CHEMISTRY. 
Relations  of  the  Carbohydrates.— Co;i//HHfd. 


Polyhydric        Aldehyde  Deriva- 
Alcohols.  lives. 


Ketone  Deriva- 
tives. 


Acid   Derivatives. 


Acid  Derivatives. 


C.H„0, 


^ft  *llft^^»i 


L/QrlnnV-'o 


C,H,^0. 


^<»r^ifi'^ft 


v^QnioV-'Q 


CH.,OH 

(CHbH)^ 

COOH 

CH,OH 

(CHOH), 

COOH 

CH,OH 

(CHOH), 

COOH 


COOH 

(CHOH), 

COOH 

COOH 

(CHOH)g 

COOH 

COOH 
(CHOH), 
COOH 


CARBOHYDRATES  PROPER. 

Following  the  usual  classification  we  have  then : 

jMonoses  or  monosaccharides, 
Saccharodioses  or  disaccharides, 
Saccharotrioses  or  trisaccharides, 
Polysaccharides. 

These  bodies  are  mostly  of  vegetable  origin,  but  some,  such 
as  sugar  of  milk,  are  found  in  the  animal  kingdom.  The  syn- 
thetic preparation  of  some  of  these  sugars  has  been  accom- 
plished, starting  from  either  formaldehyde  or  the  mixture 
called  above  glycerose.  When  formaldehyde,  CHoO,  is  treated 
with  lime  or  other  weak  bases  it  polymerizes  or  condenses 
to  a  mixture  of  sugars,  one  of  which  has  been  isolated  in 
pure  condition  and  is  known  as  a-acrose.  By  the  condensa- 
tion of  the  mixture  of  glyceraldehyde  and  dioxy-acetone  (gly- 
cerose or  triose)  mentioned  in  the  table  above  the  same  acrose 
has  been  obtained.  This  acrose  is  identical  with  the  sugar 
mixture  known  as   (d  -{-  /) -fructose. 

A  number  of  sugars  have  also  been  obtained  by  a  general 
method  of  synthesis  which  depends  on  the  fact  that  as  alde- 
hydes and  ketones  they  have  the  power  to  unite  with  hydro- 
cyanic acid  and  produce  nitriles  of  acids  which  may  be  reduced 
to  new  aldehydes  with  a  larger  number  of  carbon  atoms  than 
the  original  substance  contained.  This  may  be  illustrated  by 
starting  with  arabinose,  CgHji.Og,  as  figured  above.  This 
with  hvdrocvanic  acid  vields  a  cvanide  as  follows : 


CARBOHYDRATES    AND    RELATED    BODIES.  25 

CH.OH.(CHOH)3CHO  +  HCN  =  CH.OH.(CHOH)3.CHOHCN, 
and  this  by  the  usual  reaction  gives  arabinose  carboxyhc  acid : 

CH^OH.  (CH0H)3CH0HCN  +  2H,0  = 

CH.:0H.(CH0H)3.CH0H.C00H  +  NH3. 

By  loss  of  water  from  this  acid  the  corresponding  lactone  is 
formed : 

CHcOH.  ( CHOH)  4COOH  —  H=0  = 

CH.OH.CHOH.CH.CHOH.CHOH.CO  =  CeHioOo. 
i o 

By  reduction  with  sodium  amalgam  this  lactone  becomes  a 
sugar,  identical  with  that  obtained  by  the  other  condensation : 

CH.OH.CHOH.CH.CHOH.CHOH.CO  +  H.  = 

O 

CH.OH.  ( CHOH )  4.CHO  =  CcHi^Oe. 

By  an  extension  of  the  principle,  sugars  with  7,  8  and  9  carbon 
atoms  have  been  obtained.  In  what  is  to  follow  a  brief  dis- 
cussion of  the  more  important  natural  substances  will  be  given. 

THE  MONOSES  OR  MONOSACCHARIDES. 

A  number  of  pentose  and  hexose  bodies  must  be  considered 
here. 

Pentoses.  Small  amounts  of  these  sugar-like  compounds 
exist  in  nature,  but  they  are  mostly  derived  from  simple  ante- 
cedent substances  called  pentosans.  The  pentoses  bear  the 
same  relation  to  the  pentosans  that  dextrose  bears  to  starch; 
by  hydration   the  latter   compounds   are   converted   into  the 

former,  thus : 

C5H8O4  +  H2O  =  C5H10O5. 

QHioOs  +  H2O  =  CeHi^Oe. 

Among  the  pentoses  two,  known  as  /-arabinose  and  /-xylose, 
are  the  most  important. 

Arabinose,  or  pectin  sugar,  is  made  by  warming  cherry 
gum,  wheat  bran,  gum  arable,  quince  or  gedda  mucilage,  ex- 
hausted brewers'  grains  and  various  other  substances  with 
dilute   acids.     It   has    a    specific   rotation    [a]^  = -|- 104.5°. 


26  PHYSIOLOGICAL    CHEMISTRY. 

WHien  boiled  with  dilute  hydrochloric  acid  it  yields  furfiiral- 
dehyde. 

Xylose,  or  wood  sugar,  is  obtained  by  boiling"  wood  gum 
with  dilute  sulphuric  acid.  Its  specific  rotation  is  +  19.4°- 
Like  the  preceding  body  it  is  a  reducing  sugar,  but  non- 
fermentable.  The  nutritive  value  of  these  substances  for  man 
is  low,  but  for  the  herbivora  these  and  the  antecedent  pen- 
tosans are  more  important,  as  they  appear  to  be  rather  easily 
digested  in  the  alimentary  tract  of  many  animals.  Traces  of 
pentoses  have  occasionally  been  found  in  human  urine. 

All  these  bodies  are  distinguished  from  the  sugars  proper 
by  yielding  relatively  large  quantities  of  furfuraldehyde  when 
distilled  with  hydrochloric  acid  or  sulphuric  acid,  which  reac- 
tion may  be  illustrated  by  the  following  test : 

Ex.  In  a  small  retort  or  flask  fitted  with  a  delivery  tube  mix  5  gm.  of 
bran  and  100  cc.  of  ten  per  cent  hydrochloric  acid.  Connect  the  retort 
or  flask  with  a  condenser,  apply  heat  and  distil  over  about  half  the  liquid. 
With  a  few  drops  of  this  make  the  Schiflf  furfuraldehyde  test.  Moisten 
a  small  strip  of  paper  with  aniline  acetate  obtained  by  mixing  equal  vol- 
umes of  glacial  acetic  acid  and  aniline.  Touch  this  test-paper  with  a  glass 
rod  holding  a  drop  of  the  bran  distillate.  A  bright  red  color  appears,  due 
to  the  formation  of  furoaniline,  C4H30.CH.(C6H4NH:)2.  The  reaction  is 
extremely  delicate  and  serves  for  the  detection  of  traces  of  the  products 
yielding  furfuraldehyde,  GH3O.CHO. 

The  Hexoses.  These  are  important  substances  repre- 
sented by  the  general  formula  CfjHioOc,.  Several  occur 
widely  distributed  in  nature,  being  found  in  ripe  fruits  and 
elsewhere.  A  few  are  artificial  products  formed  by  laboratory 
operations.  Complex  combinations  of  these  bodies  known  as 
glucosides  are  also  common  and  are  essentially  ethereal  salts 
of  the  hexoses.  These  hexose  bodies  are  all  sweet,  soluble 
in  water,  nearly  insoluble  in  alcohol  and  are  all  reducing  sub- 
stances with  oxidizing  agents  like  Fehling's  solution.  They 
undergo  fermentation  readily,  and  all  the  important  forms 
yield  alcohol  and  carbon  dioxide  under  the  influence  of  the 
yeast  organism.  Some  of  them  yield  lactic  acid  when  acted 
upon  by  the  proper  ferment.     By  partial  oxidation  they  yield 


CARBOHYDRATES    AND    RELATED    BODIES.  2/ 

monocarboxylic  acids,  such  as  mannonic  acid  or  dextronic 
acid,  and  by  the  more  pronounced  oxidation  they  are  converted 
into  dicarboxyhc  acids,  as  saccharic  or  mucic  acid. 

A  reaction  of  great  importance  in  connection  with  the 
hexoses  is  that  which  they  exhibit  when  acted  upon  by  phenyl 
hydrazine.  While  this  is  a  general  aldehyde  or  ketone  reac- 
tion, the  behavior  of  the  hexoses  is  so  characteristic  as  to 
require  mention.  With  one  molecule  of  phenyl  hydrazine, 
CgHgNH  —  NH2,  the  hexoses  yield  hydrazones ,  CgHigOg 
—  N  —  NH.CgHs,  the  ketone  or  aldehyde  oxygen  being 
replaced  by  the  hydrazine  group.  The  hydrazones  are  mostly 
soluble  in  water.  An  excess  of  phenyl  hydrazine,  enough  to 
give  two  molecules  of  that  substance  to  one  of  the  hexose, 
yields  bodies  called  osasones,  which  are  mostly  yellow,  insol- 
uble crystalline  compounds  of  great  importance  for  the  sepa- 
ration and  identification  of  several  of  the  sugars.  They  are 
represented  by  the  formula  C6HioOi(N  —  NH.CeH5)2,  and 
by  warming  with  strong  hydrochloric  acid  they  yield  peculiar 
compounds  called  osones,  which  are  mixed  ketone  and  alde- 
hyde structures.     These  reactions  will  all  be  illustrated  below. 

Glucose  (J-glucose),  known  also  as  dextrose,  grape  sugar, 
or  diabetic  sugar,  is  the  best  known  representative  of  the 
group.  It  is  found  in  honey  and  many  fruit  juices,  often 
associated  with  levulose  or  fruit  sugar.  It  may  be  produced 
by  the  action  of  weak  acids  on  cellulose  or  starch  and  on  the 
large  scale  is  so  made  from  the  latter  substance.  Weak  sul- 
phuric acid  is  commonly  employed  and  the  hydration  or  con- 
version is  effected  under  pressure.  Hydrochloric  acid  is 
sometimes  employed  in  making  a  commercial  glucose,  while 
with  other  acids  the  action  is  much  slower  or  less  complete. 
In  the  section  below  on  the  behavior  of  starch  the  nature  of 
the  reaction  will  be  explained.  The  following  experiment 
will  illustrate  the  production  of  the  sugar  by  the  aid  of  sul- 
phuric acid : 

Ex.  Make  a  paste  by  boiling  about  a  gram  of  starch  with  100  cc.  of 
water  in  a  glass  flask.     Add  10  drops  of  dilute  sulphuric  acid  (1:5)   and 


25  PHYSIOLOGICAL    CHEMISTRY. 

boil  five  to  ten  minutes.  Now  allow  the  liquid  to  cool,  remove  5  cc.  with 
a  pipette,  dilute  this  to  25  cc.  with  water  and  add  a  few  drops  of  iodine 
solution ;  a  blue  violet  color  results,  showing  that  starch  or  a  starch-like 
substance  is  still  present.  The  remainder  of  the  acid  liquid  in  the  flask 
is  next  boiled  steadily  for  one  hour,  a  little  water  being  added  from  time 
to  time  to  replace  that  lost  by  evaporation.  At  the  end  of  an  hour  remove 
5  cc,  dilute  and  test  with  iodine  solution  as  before.  The  characteristic 
starch  reaction  is  now  absent,  while  the  liquid  has  become  thin  and  trans- 
parent. 

Neutralize  the  free  sulphuric  acid  by  addition  of  a  slight  excess  of  chalk 
or  fine  marble  dust,  heat  gently  to  complete  the  reaction.  Then  filter  and 
evaporate  the  filtrate  nearly  to  dryness  on  a  water-bath.  Allow  to  cool 
and  notice  that  the  residue  has  a  sweet  taste.  It  is,  in  fact,  glucose  and 
the  experiment  illustrates  the  method  of  manufacturing  glucose  on  the 
large  scale.  Test  it  by  dissolving  a  little  in  water  and  adding  a  few  drops 
of  Fehling's  solution,  described  below.  On  boiling,  a  yellowish  precipitate 
appears  which  becomes  bright  yellow  and  finally  red. 

Under  certain  conditions  it  is  possible  to  obtain  a  pure 
crystalline  product  from  the  syrup  made  as  just  illustrated. 
This  is  known  as  crystallized  grape  sugar  or  pure  anhydrous 
dextrose.  The  common  commercial  product,  sold  as  glucose 
syrup,  often  contains  much  unconverted  .  dextrin  from  the 
incomplete  hydrolysis  of  the  starch.  At  the  present  time  large 
quantities  of  glucose,  both  solid  and  liquid,  are  made  and  used 
in  the  fermentation  industries,  by  bakers  and  confectioners 
and  in  the  household.  Glucose  is  sweet,  but  not  as  sweet  as 
cane  sugar,  and,  because  of  the  fact  that  it  readily  undergoes 
fermentation,  it  can  not  replace  cane  sugar  for  certain  pur- 
poses, such  as  the  preparation  of  the  syrups  of  the  pharma- 
copoeia or  the  canning  or  preserving  of  fruits. 

The  typical  aldose  reactions  are  well  shown  with  a  solution 
of  glucose.  For  the  first  of  these  we  require  a  reagent,  re- 
ferred to  above  as  Fehling's  solution,  which  is  made  as 
follows : 

Fehling's  Solution.  Ex.  Dissolve  69.28  gm.  of  pure  crystallized  copper 
sulphate  in  distilled  water  and  dilute  to  make  a  liter  of  solution.  In  a 
second  portion  of  distilled  water  dissolve  100  gm.  of  pure  solid  sodium 
hydroxide  and  350  gm.  of  pure  recrystallized  Rochelle  salt  by  aid  of  heat. 
Cool  and  dilute  to  make  one  liter.  These  solutions  when  mixed  yield  the 
Fehling's  solution  proper.  It  is  best  to  mix  equal  volumes,  quite  accurately 
measured,  just  before  the  reagent  is  needed  for  use. 


CARBOHYDRATES    AND    RELATED    BODIES.  29 

This  Fehling's  solution  is  commonly  employed  as  a  quali- 
tative test  for  reducing  sugars  in  general,  but  the  reaction 
may  be  first  illustrated  by  a  simpler  method : 

Ex.  Trommer's  Test.  Add  to  a  dilute  solution  of  glucose  a  consid- 
erable excess  of  strong  potassium  hydroxide  solution  and  then  a  very  few 
drops  of  a  dilute  copper  sulphate  solution.  This  produces  no  precipitation 
but  imparts  a  deep  blue  color.  On  warming  the  solution  a  yellowish  pre- 
cipitate forms,  which  grows  bright  red  by  boiling.  This  is  cuprous  oxide, 
and  the  test  is  known  as  "  Trommer's "  test.  It  is  frequently  employed 
to  detect  the  pre'sence  of  sugar  in  liquids,  especially  in  urine,  but  on  the 
whole  is  not  as  satisfactory  as  the  next  one. 

Ex.  Fehling's  Test.  To  a  very  weak  glucose  solution  add  an  equal 
volume  of  diluted  Fehling's  solution.  The  mixture  remains  deep  blue  in 
the  cold,  but  on  heating  a  yellowish  precipitate  turning  to  red  is  soon  pro- 
duced. This  precipitate  of  cuprous  oxide  comes  from  the  reduction  of  the 
cupric  compound  held  in  solution  in  the  test  reagent.  In  the  first  or  Trom- 
mer  test  the  first  indication  of  the  presence  of  a  sugar  is  the  formation  of 
a  deep  blue  clear  solution  rather  than  a  greenish  blue  precipitate  of  cupric 
h3'droxide  which  would  result  from  the  action  of  the  copper  sulphate  and 
alkali  alone.  But  cupric  hydroxide  dissolves  in  solutions  of  sugars  and 
other  polyhydric  alcohols,  the  solution  being  deep  blue,  and  stable  in  the 
cold.  In  the  case  of  glucose  and  other  reducing  (aldehyde  or  ketone) 
sugars  this  stability  is  only  temporary,  since  reduction  follows  on  boiling. 
If  the  pol3^hydric  alcohol  employed  to  produce  the  deep  blue  solution  is 
not  a  reducing  substance  the  liquid  remains  clear  and  stable,  even  on  boil- 
ing. This  is  the  case  with  the  Fehling's  solution,  in  which  the  cupric 
hydroxide  is  held  dissolved  through  the  alcoholic  behavior  of  the  Rochelle 
salt.  Similar  solutions  are  made  by  the  aid  of  glycerol  (trihj-dric)  and 
mannitol  (hexahydric).  The  Fehling  test  has  this  advantage  over  the 
Trommer  test,  that  in  the  latter  if  too  much  copper  sulphate  is  used,  and 
little  sugar  is  present  the  precipitate  on  boiling  may  be  mainly  black  cupric 
oxide  instead  of  the  red  cuprous  oxide.  With  the  Fehling  liquid  no  black 
precipitate  can  form. 

Ex.  Bismuth  Reduction  Test.  Add  to  a  glucose  solution  some  strong 
potassium  hydroxide  solution  and  then  a  very  small  amount  of  bismuth 
subnitrate.  For  an  ordinary  test  a  few  milligrams  will  be  enough.  On 
boiling,  a  black  precipitate  appears,  which  frequently  forms  a  bright  mir- 
ror on  the  walls  of  the  test-tube.  This  precipitate  seems  to  be  a  mixture 
of  metallic  bismuth  with  some  oxide,  and  shows  the  strong  reducing  power 
of  the  sugar.  Similar  reductions  may  be  obtained  from  alkaline  solutions 
of  several  heavy  metals,  but  these  tests  illustrate  the  general  principle. 

Another  test  which  serves  for  the  recognition  of  even  minute 
traces  of  glucose  and  other  sugars,  is  the  following,  proposed 
by  Molisch : 


30  PHYSIOLOGICAL    CHEMISTRY. 

Ex.  To  a  small  amount  of  a  dilute  sugar  solution  add  two  drops  of  a 
solution  of  a-naphthol,  containing  about  20  gm.  in  100  cc.  On  shaking  the 
liquid  becomes  turbid.  Now  add  to  it  an  equal,  or  slightly  greater,  volume 
of  pure  strong  sulphuric  acid  and  shake.  A  deep  violet  color  appears, 
which  gives  place  to  a  violet  precipitate  on  addition  of  water.  This  reac- 
tion has  been  shown  to  be  due  to  the  combination  of  the  a-naphthol  with 
furfuraldehyde  produced  by  the  action  of  sulphuric  acid  on  the  sugar 
present. 

Ex.  Phenyl  Hydrazine  Test.  A  characteristic  reaction  of  great  prac- 
tical value,  referred  to  above,  is  given  on  the  addition  of  phenyl  hydrazine 
to  a  solution  of  glucose  under  certain  definite  conditions. 

To  20  cc.  of  a  dilute  glucose  solution  add  about  a  gram  of  phenyl 
hydrazine  hydrochloride,  and  two  grams  of  sodium  acetate.  Heat  on  the 
water-bath  half  an  hour,  and  then  allow  the  liquid  to  cool.  There  will  now 
be  found  a  beautiful  yellow  crystalline  precipitate  of  phenyl  glucosazone, 
the  nature  of  which  is  best  seen  under  the  microscope.  This  test  is  one 
of  great  delicacy,  and  has  been  applied  to  the  detection  of  traces  of  sugar 
in  urine.  But  care  must  be  taken  to  keep  the  reagent  in  considerable  ex- 
cess, since  otherwise  the  soluble  hydrazone  may  be  formed.  The  melting 
point  of  the  pure  osazone  is  205°  C.  The  following  reactions  illustrate  the 
combination  of  the  phenyl  hydrazine : 

CcHi.Oe  +  C0H5.NH.NH2  =  CeH.^Oj.N.NH.CeH,  -f-  H.O, 

GH,=Oo  +  2C6H5NH.NH=  =  GHaoO^.  (N.NH.CH,)^  +  2H.O  +  H=, 

Phenyl  glucosazone 

CeHvNH.NH.  -f  H.  =  CoH.NHc  +  NH^. 

By  treatment  with  strong  hydrochloric  acid  the  osazone  decomposes  to 
yield  an  osone  and  phenyl  hydrazine  hydrochloride : 

CcHioO...('N.NH.C6HO=-|-  2HCI  +  2H.O  = 

2C0H5.NH.NH..HCI  -\-  CH.OH.  (CHOH)n.CO.CHO. 

Glucosone 

This  osone  on  reduction  with  nascent  hydrogen  yields  a  sugar,  not  glu- 
cose, but  rf-fructose,  or  levulose : 

CH.OH.(CHOH)3.CO.CHO  +  H.  =  CH.OH(CHOH)3.CO.CH:OH. 

Glucose   and   levulose    (d-fructose)    yield   the   same  glucosazone;    we   are 
therefore  able  by  this  reaction  to  pass  from  one  sugar  to  the  other. 

The  ready  fermentation  of  glucose  will  be  shown  later  in  the 
discussion  of  fermentation  reactions  in  general.  The  produc- 
tion of  glucose  from  cane  sugar  will  also  be  explained.  The 
specific  rotation  of  glucose  in  20  per  cent  solution  is  given 
by  the  formula  [a]^  =53°,  and  increases  slightly  with  the 
concentration. 


CARBOHYDRATES    AND    RELATED    BODIES.  31 

(i-pRUCTOSE,  fruit  sugar,  levulose,  is  a  ketohexose  similar 
to  c^-glucose  in  some  respects  but  very  different  in  others.  It 
occurs  in  honey  and  sweet  fruits,  but  is  not  easily  separated 
in  a  pure  state  because  it  is  very  soluble  and  does  not  readily 
crystallize.  The  preparation  of  glucose  from  ordinary  starch 
has  been  referred  to  above.  In  like  manner  fructose  may  be 
obtained  from  certain  less  common  starches,  especially  from 
inulin  by  hydrolysis  with  very  weak  acid.  In  pure  condition 
this  sugar  has  no  technical  importance. 

The  various  reduction  and  fermentation  reactions  are 
shown  by  fructose  as  well  as  by  glucose,  but  the  quantitative 
relation  between  copper  hydroxide  and  fructose  is  not  quite 
the  same  as  with  glucose.  As  they  yield  the  same  osazone 
the  phenyl  hydrazine  test  can  not  be  employed  to  distinguish 
between  them.  The  most  characteristic  property  of  fructose 
is  found  in  its  optical  behavior.  While  the  specific  rotation 
of  J-glucose  is  about  53°  to  the  right,  that  of  ^/-fructose  is, 
at  20°  C,  and  for  a  strength  of  20  per  cent,  about  93°  to 
the  left.  Because  of  this  behavior  the  sugar  is  commonly 
called  levulose.  Another  reaction  which  may  be  applied  is 
this: 

Ex.  Dissolve  resorcin  in  20  per  cent  hydrochloric  acid  and  heat  a  lit- 
tle of  this  solution  with  the  levulose  solution  to  be  tested.  A  red  color 
results.  At  the  same  time  a  precipitate  forms  which  may  be  dissolved  in 
alcohol  with  a  red  color.  One-tenth  gram  of  resorcin  to  5  cc.  of  the  acid 
is  sufficient. 

J-Galactose.  This  is  the  third  hexose  of  importance,  but 
it  is  not  a  natural  substance.  In  the  inversion  of  milk  sugar 
by  weak  acids  galactose  is  formed  along  with  glucose,  and  it 
results  also  from  the  action  of  acids  on  several  gums.  The 
sugar  is  readily  soluble  in  water,  fermentable  and  dextro- 
rotatory like  glucose.  It  forms  a  characteristic  osazone.  It 
reduces  Fehling's  solution  but  not  in  the  same  proportion  as 
glucose.  On  reduction  it  yields  dulcitol,  which  shows  its 
chemical  relations  most  characteristically.  On  oxidation  it 
yields  galactonic  and  mucic  acids. 


32  PHYSIOLOGICAL    CHEMISTRY. 

^-Talose  is  an  unimportant  aldose  of  artificial  origin. 

Sorbinose  is  a  ketone  sugar  obtained  from  the  juice  of  the 
mountain  ash  berry.  It  is  levorotatory  and  non-fermentable 
with  yeast. 

Invert  Sugar.  This  name  is  given  technically  to  the  mix- 
ture of  glucose  and  fructose,  equal  molecules,  produced  by  the 
action  of  weak  acids  on  cane  sugar,  as  described  below. 

THE    CANE    SUGAR    GROUP. 

The  Saccharobioses  or  Disaccharides.  These  are  sugars 
of  the  formula  CioHgoOu  and  are  important  substances. 
The  best  known  representatives  of  the  class  are  cane  sugar, 
milk  sugar  and  malt  sugar,  all  of  which  are  natural  products, 
with  cane  sugar  the  most  abundant.  These  bodies  are  closely 
related  to  the  hexoses,  two  molecules  of  the  latter  being  in 
some  manner  condensed  or  united  to  produce  one  of  the 
former.  The  disaccharides,  on  the  other  hand,  by  treatment 
with  weak  acids  or  certain  ferments  break  up  easily  into  two 
molecules  of  a  hexose,  water  being  added  in  the  reaction : 

Cr.H.,On  +  H:0  —  CoHi^Oc  +  CeH^Oc. 

The  hexose  molecules  formed  may  be  alike  or  different,  and 
the  process  of  converting  the  disaccharides  into  monosaccha- 
rides in  this  manner  is  called  "  inversion."  By  this  inversion 
the  following  changes  should  be  noted : 

Saccharose  or  cane  sugar  yields  glucose  and  fructose. 

Lactose  or  milk  sugar  yields  glucose  and  galactose. 

Maltose  or  malt  sugar  yields  glucose  and  glucose. 

Saccharose.  This  sugar  has  been  known  from  earliest 
times  to  some  peoples,  but  did  not  become  an  article  of  com- 
merce until  after  the  discovery  of  the  Americas.  It  is  found 
in  the  juices  of  various  canes,  several  kinds  of  beets,  the  saps 
of  many  trees  and  in  many  seeds  and  nuts.  On  the  com- 
mercial scale  it  is  produced  from  the  beet  and  canes  and  in 
smaller  amount  from  maple  sap. 

Cane  sugar  does  not  undergo  fermentation  directly  with 


CARBOHYDRATES    AND    RELATED    BODIES.  33 

pure  yeast,  but  by  prolonged  action  of  common  yeast  on  a 
dilute  solution  of  the  sugar  fermentation  appears.  This  is 
due  to  the  fact  that  the  crude  yeast  contains  an  inverting 
enzyme  known  as  invertase  which  produces  glucose  and  fruc- 
tose which  then  yield  to  the  true  fermentation.  Cane  sugar 
gives  no  combination  with  phenyl  hydrazine,  and  is  not  a 
reducing  sugar.  These  facts  point  to  the  absence  of  aldehyde 
or  ketone  groups  in  the  large  molecule.  An  experiment  illus- 
trates this : 

Ex.  Prepare  a  dilute  solution  of  pure  cane  sugar  and  boil  it  with 
Fehling  solution  in  the  usual  manner.  Observe  that  no  reduction  of  the 
copper  compound  takes  place.  Next  boil  a  similar  cane  sugar  solution 
with  a  few  drops  of  dilute  hydrochloric  or  sulphuric  acid  several  minutes, 
neutralize  with  sodium  carbonate,  and  then  apply  the  Fehling  test.  The 
characteristic  red  precipitate  now  appears.  In  this  reaction  the  cane 
sugar  is  broken  up  by  the  acid  into  a  molecule  of  glucose,  and  a  molecule 
of  fructose,  both  reducing  sugars  as  explained  above. 

The  behavior  of  cane  sugar  solutions  with  polarized  light 
is  characteristic  and  affords  the  simplest  and  most  accurate 
means  for  quantitative  determination.  The  specific  rota- 
tion is  practically  independent  of  the  concentration  and  is  rep- 
resented by  the  formula   [a]  ^=  -\-  66.^°. 

Strong  solutions  of  cane  sugar,  "  syrups,"  are  used  in  the 
household  and  in  pharmacy  to  prevent  fermentation.  Hence 
the  use  of  this  sugar  in  the  canning  or  preserving  of  fruit. 

Lactose.  This  is  the  characteristic  sugar  of  all  kinds  of 
milk,  with  possibly  one  or  two  exceptions.  It  may  be  sepa- 
rated from  the  "  whey  "  which  is  the  product  remaining  after 
skimming  and  precipitating  the  casein.  It  is  made  commer- 
cially in  large  quantities  as  a  by-product  in  the  cheese  industry, 
and  in  pure  crystallized  form  has  the  formula  C12H22O11.H2O. 

Milk  sugar  resembles  cane  sugar  in  respect  to  the  condi- 
tions under  which  it  may  be  fermented,  but  it  is  a  reducing 
sugar  directly,  acting  strongly  on  copper  or  bismuth  solutions. 
In  its  behavior  with  polarized  light  it  resembles  glucose 
closely,  having  a  specific  rotation,  [a]^=  -^  52.5°.  Inverted 
milk  sugar  ferments  readily,  and  products  known  as  kumyss, 
4 


34  PHYSIOLOGICAL    CHEMISTRY. 

from  mare's  milk,  and  kcphir,  from  cow's  milk,  are  made  in 
this  ^vay.  In  digestion  lactose  splits  up  into  glucose  and 
galactose  readily,  while  cane  sugar  yields  glucose  and  fructose, 
but  less  readily.  With  phenyl  hydrazine  a  yellow  lactosazone 
is  formed. 

Milk  sugar  is  much  less  soluble  in  water  than  is  cane  sugar 
and  has  but  a  slightly  sweet  taste.  It  is  used  mainly  in  the 
production  of  infant  and  invalid  foods  and  in  manufacturing 
pharmacy  in  tablets,  pills,  etc. 

Maltose,  or  the  sugar  of  malt,  is  produced  by  the  action 
of  malt  diastase  on  starch.  It  therefore  occurs  in  germinating 
seeds  and  grains,  and  is  present  wherever  a  diastase  acts  on 
starch.  In  the  action  of  weak  acids  on  starch  paste  malt  sugar 
is  produced  as  a  transition  stage,  glucose  finally  resulting  by 
inversion.  In  this  countr\'  malt  sugar  is  not  a  common  article 
of  commerce,  but  in  several  European  countries  it  has  been 
produced  in  considerable  quantities  to  be  used  as  an  article 
of  food  in  the  place  of  glucose  or  cane  sugar.  The  manufac- 
ture of  a  relatively  pure  sugar  b}'  the  use  of  malt  diastase  and 
a  starchy  material,  such  as  corn,  seems  to  be  attended,  how- 
ever, with  great  practical  difficulties. 

]Maltose  is  readily  soluble  in  water,  sweet,  but  not  to  the 
same  degree  as  cane  sugar,  and  is  not  directly  fermentable. 
But  an  inverting  enzyme  in  common  yeast  changes  it  so 
quickly  that  it  was  long  classed  among  the  true  fermenting 
sugars.  The  view  is  now  generally  held  that  the  disaccha- 
rides  must  first  be  converted  into  monosaccharides  before  real 
fermentation  can  take  place.  In  the  industries  malt  sugar 
is  thus  fermented  on  the  large  scale.  Toward  oxidizing  solu- 
tions its  behavior  is  like  that  of  glucose,  although  its  reducing 
power  is  not  quite  as  great.  \Mth  phenyl  hydrazine  it  forms 
a  maltosazone,  and  on  polarized  light  its  rotating  power  is  very 
great,  the  specific  rotation  being  at  20°  C.  {a]j^,z=-\-  137^. 
In  the  body  maltose  is  changed  into  glucose  by  action  of  an 
inverting  enzyme  occurring  both  in  the  pancreas  and  in  the 
true  intestinal  juice.     This  inversion  seems  to  take  place  much 


CARBOHYDRATES    AND    RELATED    BODIES.  35 

more  readily  than  in  the  case  of  cane  sugar,  which  is  a  fact 
of  considerable  physiological  importance. 

IsoMALTOSE.  This  is  a  sugar  which  has  been  made  by  the 
action  of  fuming  hydrochloric  acid  on  glucose.  It  also  accom- 
panies the  true  maltose  in  the  products  formed  by  the  action 
of  diastase  on  starch.  It  differs  from  maltose  in  rotating 
power,  which  is  much  less,  in  the  character  of  its  phenyl  osa- 
zone,  and  in  water  solubility.  It  reduces  copper  and  bismuth 
solutions  but  undergoes  fermentation  with  yeast  very  slowly. 

Other  disaccharides  known  have  but  little  importance.  Mycosc  or 
trehalose  is  found  in  certain  fungi,  agarose  is  obtained  from  the  juice 
of  the  agave  plant.  Melibiose  and  turanose  are  formed  in  the  hydrolysis 
of  certain  polysaccharides. 

The  Saccharotrioses  or  Trisaccharides.  This  group  con- 
tains a  few  sugars  and  but  one  of  these  is  important  at  the 
present  time. 

Melitose  or  raffinose.  This  sugar,  having  the  formula 
CigHsaOie .+  5H2O,  is  found  in  certain  kinds  of  manna  and 
also  in  sugar  beets  in  small  amount.  It  is  characterized  by 
having  a  strong  rotation,  [a]^  =  104.5°.  Being  more  sol- 
uble than  saccharose  it  is  found  in  the  last  crystallizations  from 
beet  juice,  and  thus  sometimes  contaminates  the  beet  sugar. 
Its  high  rotation  may  cause  an  error  in  the  estimation  of 
sugar  by  the  polarimeter.  When  inverted  with  acids  it  yields 
fructose,  and  the  disaccharide  melibiose. 

THE    DETERMINATION   OF    SUGARS. 

This  is  carried  out  in  several  ways.  In  one  method  the 
reactions  depend  on  the  reducing  power  of  sugars  on  alkaline 
copper  or  other  metallic  solutions.  The  Fehling  reagent  is 
usually  employed.  Methods  with  the  polariscope  will  be 
described  later. 

Method  with  Fehling's  Solution.  Fehling's  solution,  as 
described  above,  is  made  arbitrarily  of  such  a  strength  that  one 
cubic  centimeter  is  reduced  by  5  milligrams  of  glucose,  on  the 
supposition  that  the  sugar  and  copper  salt  react  on  each  other 


36  PHYSIOLOGICAL    CHEMISTRY. 

in  the  proportion  of  one  molecule  of  glucose  to  five  molecules 
of  crystallized  copper  sulphate. 

It  was  formerly  held  that  the  reaction  was  a  perfectly 
definite  and  simple  one,  and  could  be  expressed  in  this  manner, 
but  it  is  now  known  that  the  dilution  of  the  solutions  is  a  very 
important  factor  in  determining  the  amount  of  copper  reduced. 
The  best  conditions  to  be  employed  in  practice  have  been  deter- 
mined by  Soxhlet,  who  found  the  reducing  power  of  several 
sugars  to  vary  as  follows,  when  they  were  tested  in  solutions 
of  I  per  cent  strength : 

0.5  gm.  of  invert  sugar  in  i  per  cent  solution  reduces  101.2  cc.  of 
Fehling's  solution,  undiluted. 

0.5  gm.  of  invert  sugar  in  I  per  cent  solution  reduces  97.0  cc.  of 
Fehling's  solution,  diluted  with  4  volumes  of  water. 

0.5  gm.  of  glucose  in  i  per  cent  solution  reduces  105.2  cc.  of  Fehling's 
solution,  undiluted. 

0.5  gm.  of  glucose  in  i  per  cent  solution  reduces  loi.i  cc.  of  Fehling's 
solution,  diluted  with  4  volumes  of  water. 

0.5  gm.  of  milk  sugar  in  i  per  cent  solution  reduces  74  cc.  of  Fehling's 
solution,  undiluted.     The  reducing  power  in  diluted  solution  is  the  same. 

0.5  gm.  of  maltose  in  i  per  cent  solution  reduces  64.2  cc.  of  Fehling's 
solution,   undiluted. 

0.5  gm.  of  maltose  in  i  per  cent  solution  reduces  67.5  cc.  of  Fehling's 
solution,  diluted  with  4  volumes  of  water. 

The  oxidizing  power  of  i  cc.  of  Fehling's  solution  with  each  kind  of 
sugar  may  be  tabulated  as  follows,  assuming  the  sugars  to  be  in  solutions 
of  approximately  i  per  cent  strength  when  acted  upon. 

One  cubic  centimeter  of  Fehling's  solution  oxidizes : 

When  When  Diluted  with 

Undiluted.  4  Vols,  of  Water. 

Glucose 4.75  mg.  4.94  mg. 

Invert  Sugar   4.94    "  5.15     " 

Milk   Sugar    6.76    "  6.76    " 

Maltose    7.78    "  7.40    " 

The  practical  application  of  the  test  is  best  shown  by  an  experiment. 

Ex.  Measure  out  accurately  into  a  flask  holding  about  250  cc,  25  cc. 
of  the  copper  solution  and  the  same  volume  of  the  alkaline  tartrate.  Heat 
the  mixture,  or  Fehling's  solution,  on  a  wire  gauze  and  note  that  it 
remains  clear.  Fill  a  50  cc.  burette  with  a  dilute  glucose  solution  and 
run  10  cc.  into  the  hot  liquid.  Boil  two  minutes,  shaking  the  flask  con- 
tinuously, and  allow  the  mixture  to  settle.  If  the  supernatant  liquid  ap- 
pears yellow  the  mixture  indicates  that  the  sugar  solution  is  much  too 


CARBOHYDRATES    AND    RELATED    BODIES.  37 

strong  and  must  be  diluted  with  at  least  an  equal  volume  of  water  before 
beginning  another  test.  If,  on  the  other  hand,  the  liquid  is  still  blue,  add 
2  cc.  more  of  the  sugar  solution,  boil  again  for  two  minutes  and  allow 
to  settle.  If  the  color  is  now  yellow  an  approximate  value  for  the  amount 
of  sugar  in  the  solution  becomes  known,  but  if  still  blue,  the  operation  of 
adding  solution  and  boiling  must  be  continued  until,  after  settling,  a  yellow 
color  appears.  Approximately  250  mg.  of  glucose  is  required  to  reduce  the 
Fehling  solution  taken,  and  this  must  be  contained  in  the  sugar  solution 
added.  From  this  preliminary  experiment  calculate  the  amount  of  sugar 
present  in  each  cubic  centimeter. 

Ex.  With  the  data  obtained  in  the  above  experiment  as  a  basis,  make 
now  a  new  sugar  solution,  having  a  strength  of  about  i  per  cent.  Measure 
out  50  cc.  of  the  Fehling's  solution,  heat  to  boiling  and  run  the  new  sugar 
solution  from  the  burette  as  before,  the  first  addition  being  about  20  cc. 
Boil  and  note  the  color  after  settling  and  then  cautiously  continue  the 
addition  of  sugar  solution,  a  few  tenths  of  a  cc.  at  a  time,  boiling  after 
each  addition,  until  the  blue  color  gives  place  to  a  yellowish  green  and 
then,  by  the  addition  of  a  drop  or  two,  to  a  pale  j^ellow. 

Sometimes  the  final  disappearance  of  the  copper  from  the  solution  is 
determined  by  filtering  a  few  drops  through  a  very  small  filter  and  add- 
ing a  drop  of  acetic  acid  and  a  drop  of  ferrocyanide  solution  to  the  filtrate, 
when  the  characteristic  reddish  color  is  given  if  a  trace  of  copper  is  pres- 
ent. 

To  determine  cane  sugar  by  the  Fehling's  solution  it  must  first  be  con- 
verted or  "  inverted  "  into  a  mixture  of  glucose  and  fructose.  If  the  sugar 
is  in  the  dry  condition  the  inversion  can  be  accomplished  as  follows : 
Weigh  out  9.5  gm.,  dissolve  in  700  cc.  of  water,  add  20  cc.  of  normal 
hydrochloric  acid  and  heat  for  30  minutes  on  the  water-bath.  Then  neu- 
tralize with  20  cc.  of  normal  sodium  hydroxide  solution  and  make  up  to 
1000  cc.  on  cooling.  This  gives  now  a  i  per  cent  solution,  which  is  em- 
ployed as  given  for  glucose,  using  the  factor  4.94  instead  of  4.74  as  the 
amount  of  sugar  oxidized  by  each  cc.  of  the  copper  solution.  On  comple- 
tion of  the  experiment  calculate  95  parts  of  cane  sugar  for  each  100  parts 
of  invert  sugar  found. 

The  attention  of  the  student  is  directed  to  the  fact  that  malt  sugar  and 
milk  sugar  may  be  determined  by  the  aid  of  Fehling's  solution  without 
previous  inversion.     This  should  be  verified  by  experiment. 

Method  by  Use  of  Ammoniacal  Copper  Solutions.     The 

determination  of  glucose  in  pure  aqueous  solution  by  the  above 
method  is  simple  and  accurate,  but  in  liquids  containing  other 
organic  matters  the  precipitate  sometimes  fails  to^  settle  read- 
ily, so  that  the  recognition  of  the  end  point  is  difficult.  This 
is  often  the  case  with  urine  and  other  physiologically  important 
liquids.     Advantage  may  be  taken  of  the  fact  that  cuprous 


38  PHYSIOLOGICAL    CHEMISTRY. 

oxide  dissolves  in  ammonia  without  color  to  prepare  a  quan- 
titative solution  with  which  this  difificulty  may  be  largely  over- 
come. Pavy  was  the  first  to  employ  such  a  reagent  prac- 
tically and  his  solution  was  made  by  diluting  the  ordinary 
Fehling's  solution  with  ammonia  in  certain  proportion.  His 
suggestion  has  received  several  modifications.  In  all  of  these 
the  weak  sugar  solution  is  added  to  the  boiling  ammoniacal 
copper  solution  until  the  color  of  the  latter  is  just  discharged, 
at  which  point  the  reduction  of  the  copper  hydroxide  by  the 
sugar  is  complete.  In  place  of  using  the  Fehling's  solution 
it  is  well  to  make  the  Loewe  solution  with  glycerol  the  basis 
of  the  dilution.  A  solution  of  this  kind  may  be  made  by  the 
formula  below,  in  which  the  proportions  have  been  found  by 
the  present  writer  to  give  the  best  result  in  practical  work. 
One  cubic  centimeter  of  the  solution  oxidizes  one  milligram 
of  glucose  in  0.2  per  cent  solution. 

It  is  made  with  the  following  amounts  per  liter: 

Copper  sulphate,  cryst 8.166  gm. 

Sodium  hydroxide  (loo  per  cent) 15.000     " 

Glj'cerol  25.000  cc. 

Ammonia  water,  0.9  sp.  gr 350.000    " 

Water  to  make 1,000.000    " 

Ex.  Of  this  solution,  measure  50  cc.  into  a  flask  and  dilute  with  water 
to  100  cc.  To  prevent  too  rapid  an  escape  of  ammonia  and  avoid  reoxida- 
tion  to  some  e.xtent,  add  to  the  mixture,  while  warming,  enough  pure  white 
solid  paraffin  to  make  a  layer  3  or  4  millimeters  in  thickness  when  melted. 
The  burette  tip  for  discharging  the  sugar  solution  is  made  long  enough  to 
pass  down  the  neck  of  the  flask  and  below  this  paraffin.  By  boiling  gently 
and  adding  the  weak  saccharine  liquid  slowly,  very  close  and  constant  re- 
sults may  be  obtained.  At  the  end  of  the  titration  the  paraffin  is  solidified 
by  inclining  the  flask  and  immersing  it  in  cold  water,  or  by  flowing  cold 
water  over  it.  The  reduced  liquid  is  then  poured  out  and  the  cake  of 
paraffin  is  thoroughly  washed  for  the  next  test.  A  flask  so  prepared  may 
be  used  for  a  hundred  titrations.  To  prevent  bumping  and  facilitate  easy 
and  uniform  boiling,  it  is  well  to  add  a  few  ver>'  small  fragments  of  pumice- 
stone. 

A  solution  made  as  above  is  not  too  strong  in  copper  for  accurate  work, 
but  the  volume  of  ammonia  necessary  to  hold  a  much  larger  amount  of  the 
reduced  oxide  in  solution  would  render  the  process  very  inconvenient. 
Some  practice  is  necessary  to  show  just  how  fast  the  saccharine  solution 


CARBOHYDRATES    AND    RELATED    BODIES. 


39 


may  be  safely  added.     If  added  too   rapidly  the  end  point  may  be  over- 
looked and  the  sugar  content  made  to  appear  too  low. 

Polarization  Tests  and  the  Use  of  the  Polariscope.  This  is  the  proper 
place  to  show  the  applications  of  the  polariscope  in  the  examination  of 
sugars  and  other  substances.  For  a  description  of  the  various  forms  of 
polariscopes  and  discussion  of  the  optical  principles  involved  in  their  con- 


FiG.  I.  A  common  form  of  Laurent  polariscope.  The  polarizing  prism  is 
situated  in  the  tube  below  H,  the  analyzer  at  E,  B  is  the  reading  microscope 
and  C  a  vernier. 

struction  the  reader  is  referred  to  the  author's  translation  of  Landolt's 
work,  "  The  Optical  Rotation  of  Organic  Substances  and  its  Practical 
Applications,"  but  a  few  words  of  elementary  explanation  may  be  in  order 
here.  A  simple  form  of  polarimeter  in  common  use  is  shown  in  the  illus- 
trations. 

In  the  polariscopes  in  common  use  for  general  scientific  studies  homo- 
geneous yellow  light  is  employed  and  this  is  first  polarized  by  passing 
through  a  specially  designed  prism  in  the  front  part  of  the  instrument.    This 


Fig.  2.  This  represents  the  course  of  the  light  through  the  Laurent  polari- 
scope, the  direction  being  reversed,  however,  from  that  of  the  last  figure,  a 
is  a  bichromate  plate  to  purify  the  light,  h  the  polarizing  Nicol,  c  a  thin 
quartz  plate  covering  half  the  field  and  essential  in  producing  a  second 
polarized  plane,  d  the  tube  to  contain  the  liquid  under  examination,  e  the 
analyzing  Nicol  and  /  and  g  the  ocular  lenses. 


40  PHYSIOLOGICAL    CHEMISTRY. 

prism  is  usually  some  form  of  a  Nicol  prism  and  is  so  constructed  that 
only  one  of  the  polarized  rays  produced  at  the  start  is  allowed  to  emerge 
and  pass  through  the  instrument.  The  plane  in  which  this  ray  vibrates 
is  called  the  plane  of  polarization.  Such  plane  polarized  light  passes 
through  air,  water,  alcohol,  ether,  glass  and  many  other  transparent  sub- 
stances without  change;  that  is  the  direction  in  which  the  light  vibrates 
remains  unaltered.  But  many  organic  substances,  liquids  or  solids  dis- 
solved, have  the  remarkable  property  of  causing  this  plane  of  polarization 
to  change  direction;  in  other  words  the  plane  of  vibration  of  the  light  suf- 
fers a  twist  or  rotation  in  passing  through  a  column  of  the  liquids.  Sub- 
stances which  have  the  power  of  changing  the  direction  of  the  plane  of 
vibration  of  polarized  light  passing  through  them  are  called  "  active  "  sub- 
stances and  the  extent  of  the  rotation  is  dependent  on  the  number  of  mole- 
cules which  the  light  passes.  In  the  case  of  homogeneous  liquids  like  oil 
of  turpentine  the  rotation  varies  with  the  length  of  the  column  through 
which  the  light  must  pass,  while  in  the  case  of  dissolved  solids,  sugar 
solutions  for  example,  the  amount  of  the  twist  or  rotation  varies  with 
the  length  of  the  column  of  solution,  and  also  with  its  concentration  or 
number  of  molecules  in  a  given  volume.  An  instrument  which  has  some 
device  which  enables  the  observer  to  read  off  this  rotation  in  degrees  is 
called  a  polarimctcr,  and  the  number  of  degrees  read  constitutes  a  measure 
of  the  strength  or  concentration  of  the  substance. 

In  order  to  compare  the  rotation  of  substances  the  term  "  specific  rota- 
tion "  has  been  introduced.  This,  as  applied  to  liquids,  may  be  defined  as 
the  rotation  which  a  substance  would  exhibit  if  examined  in  a  column  loo 
millimeters  in  length  having  a  concentration  of  i  gram  of  active  substance 
to  each  cubic  centimeter.  This  rotation  must  therefore  be  a  calculated  one, 
and  is  found  as  illustrated  by  this  concrete  case.  Consider  a  solution 
made  by  dissolving  25  gm.  of  pure  cane  sugar  in  distilled  water  and  diluted 
to  make  exactly  100  cc.  This  is  then  examined  in  a  polarization  tube, 
which  is  a  long  tube  of  glass  or  metal  having  ends  of  plane  polished  glass 
perfectly  parallel  to  each  other.  The  sugar  solution  forms  then  a  clear 
transparent  column  of  definite  length,  which,  assume  in  this  case,  is  200 
millimeters.  By  examination  in  the  polarimeter  it  is  found  now  that  this 
solution  rotates  the  plane  of  polarized  sodium  light  through  33.25°.  For  a 
solution  with  100  grams  to  100  cc.  the  rotation  by  calculation  should  be 
four  times  this,  or  133°,  in  the  200  mm.  tube  or  66.5°  in  the  100  mm.  or 
standard  tube.  This  is  then  the  specific  rotation,  and  we  express  it  by 
the  formula : 

[a]  ^=66.5°, 

in  which  [a]  is  the  usual  symbol  for  the  specific  rotation,  and  the  D  the 
indication  that  the  observation  is  made  with  sodium  light,  a  without  the 
brackets  is  the  angle  of  rotation  as  read  off.  Remembering  the  definition 
of  specific  rotation  we  have  this  general  formula  as  applied  to  solutions : 

r  ,       100  X  looa        \o*a 


CARBOHYDRATES    AND    RELATED    BODIES.  4 1 

in  which  /  expresses  the  length  of  the  observation  tube  in  miUimeters  and 
c  the  concentration  or  strength  of  the  solution  in  grams  per  lOO  cubic 
centimeters. 

For  many  substances  this  rotation  is  so  characteristic  and  so  easily  ob- 
served that  it  constitutes  a  good  test  of  purity  or  identity.  With  the 
specific  rotation  known  the  following  relation  enables  us  to  find  the  amount 
of  active  substance  in  solution : 

IO*a 

The  following  are  some  specific  rotations  which  have  importance  from  the 
standpoint  of  physiological  chemistry,  the  temperature  being  20°  C.  in  each 
case : 

Cane  sugar,  [a]i)  =  +   66.5°  for  c=io  to  30 

Milk  sugar   (-t-HsO),  [a]c  =  4-   52.5°  c=    3  to  40 

Malt  sugar   (+H2O),  [a]j  =  + 137.0°  c=    2  to  20 

Glucose,  [a]c  =  -l-    53.0°  c  =  20 

Levulose,  [^]d  =  —   93-0°  c=ioto20 

Invert  sugar,  ['*].o  =  —   20.2°  c=:is 

The  protein  substances,  dextrin,  glycogen  and  a  number  of  other  com- 
pounds to  be  referred  to  later  have  also  a  high  rotating  power,  which 
finds  application  in  investigations. 

THE    POLYSACCHARIDES. 

We  have  here  a  very  important  group  of  bodies,  some  of 
which  appear  to  have  an  extremely  complex  structure.  For- 
merly these  compounds  were  assumed  to  be  simpler  than  the 
sugars  and  were  represented  by  the  general  formula  CqHj^qO^. 
The  action  of  water  in  producing  glucose  was  assumed  to  con- 
sist merely  in  the  addition  of  one  molecule  as  shown  by  the . 
formula : 

But  this  view  is  no  longer  held ;  the  starches,  cellulose  bodies 
and  certain  gums  belonging  to  the  group  have  been  shown  to 
exist  in  the  form  of  large  and  probably  very  complex  mole- 
cular aggregations,  and  the  formula  (CeHjoOg),,  is  now 
usually  employed  to  indicate  this  fact. 

These  polysaccharides  are  related  to  the  real  sugars  by  sev- 
eral reactions.  By  certain  treatment  most  of  them  may  be 
converted  more  or  less  readily  into  maltose,  glucose  or  fruc- 


42 


PHYSIOLOGICAL    CHEMISTRY. 


tose.  and  besides  this  they  yield  the  ester  derivatives  charac- 
teristic of  polyhydric  alcohols.  In  their  natural  condition  they 
are  mostly  insoluble  in  water  and  other  solvents.  It  is  cus- 
tomary to  make  three  classes  of  these  compounds,  of  which 
the  starches  or  amyloses,.as  the  most  important,  will  be  treated 
first. 

The  Amyloses.  In  the  vegetable  kingdom  starch  is  a  com- 
mon and  widely  distributed  reserve  material,  a  sugar,  probably 
saccharose,  being  first  formed  as  a  synthetic  product.     Starch 


Fig.  3.     Wheat   starch   magnified   about   350   diameters. 


is  found  in  many  seeds,  grains  and  tubers  in  the  form  of 
minute  granules  which  are  often  characteristic  in  shape  or 
size.  They  may  be  extremely  small,  pepper  starch  for  ex- 
ample, or  relatively  large,  as  in  the  case  of  arrowroot  starch. 
Under  the  microscope  these  granules  often  appear  to  be  built 
up  of  concentric  layers,  and  furthermore  they  are  not  homo- 
geneous in  composition.  The  outer  part  of  the  granule  con- 
sists of  a  covering  or  sheath  of  starch  cellulose  within  which 
is  the  large  mass  of  starch  ^ranulose.     The  cellulose  sheath 


CARBOHYDRATES    AND    RELATED    BODIES.  43 

is  insoluble  in  water  at  the  ordinary  temperature,  but  with 
elevation  of  temperature  in  presence  of  an  excess  of  water  the 
protective  layer  breaks  and  allows  the  granulose  to  form  a 
more  or  less  perfect  solution  of  so-called  soluble  starch. 

On  the  technical  scale  starch  may  be  obtained  from  a  variety 
of  substances.  The  common  sources  are  potatoes,  corn,  rice 
and  arrowroot.  The  manufacture  is  largely  a  mechanical 
operation,  which  may  be  illustrated  as  follows : 

Ex.  Grate  a  potato  to  a  pulp  by  means  of  an  ordinary  tin  grater,  mix 
the  pulp  with  water  and  squeeze  through  a  piece  of  coarse  unbleached 
muslin,  collecting  the  strained  liquids  in  a  large  beaker.  Allow  the  mix- 
ture to  settle  a  half  hour  or  longer  and  pour  off  the  water,  which  contains 
some  soluble  albuminous  substances,  some  cellular  floating  matter,  but  very 
little  starch.  Most  of  this  will  be  found  in  the  bottom  of  the  beaker. 
Add  some  fresh  water,  stir  up  and  allow  to  settle.  Now  pour  the  water 
off  again  and  repeat  these  operations  until  the  starch  appears  perfectly 
clean  and  white.  Transfer  this  starch  to  a  clean  shallow  dish  and  allow 
what  is  not  intended  for  immediate  use  to  dry  spontaneously  in  an  atmos- 
phere free  from  dust.  The  dried  product  will  consist  of  minute  glistening 
particles  resembling  small  crystals.     Save  this  starch  for  tests  given  below. 

Ex.  Examine  starch  from  several  sources  under  the  microscope,  em- 
ploying a  power  of  about  300  diameters.  Clean  a  glass  slide  thoroughly, 
place  in  the  center  of  it  a  small  drop  of  water,  and  stir  into  this  by  means 
of  a  needle,  or  glass  rod,  a  minute  quantity  of  starch.  Now  drop  on  a 
clean  cover  glass  in  such  a  manner  as  to  exclude  air  bubbles,  and  place 
under  the  microscope  for  observation. 

Ex.  Repeat  the  last  experiment,  using  an  aqueous  solution  of  iodine 
instead  of  water.  The  starch  granules  will  now  appear  blue.  For  the 
detection  of  starch  in  mixtures  the  use  of  iodine  is  often  indispensable. 

Some  idea  of  the  size  of  the  starch  cells  may  be  obtained 
from  this  table  which  gives  the  mean  diameter  in  fractions  of 
a  millimeter.  For  starch  granules  which  are  oval  instead  of 
circular,  the  averages  of  the  longer  and  shorter  diameters  is 
given : 

Potato    0.06  -o.  10 

Common   arrowroot    o.oi  -0.07 

Corn    0.007-0.02 

Wheat    0.002-0.05 

Rice    0.005-0.008 

Pea    0.016-0.028 

Bean    0.035 

Barley    0.013-0.040 

Rye 0.002-0.038 


44  PHYSIOLOGICAL    CHEMISTRY. 

Starch  may  be  recognized  by  a  number  of  chemical  tests,  the  best  of 
which  are  the  following: 

Ex.  Boil  a  small  amount  of  starch  with  water  so  as  to  make  a  thin 
paste.  Allow  this  to  cool,  and  add  a  few  drops  of  an  aqueous,  or  alcoholic 
solution,  of  iodine.  A  deep  blue  color  is  formed,  which  disappears  on 
boiling  the  mixture.  This  test  is  exceedingly  delicate  and  characteristic, 
and  serves  for  the  detection  of  minute  traces  of  iodine  as  well  as  starch. 
The  blue  color  is  destroyed  by  alkalies  or  much  alcohol  as  well  as  by 
heat. 

Ex.  That  starch  is  insoluble  in  cold  water  may  be  shown  by  stirring 
some  with  water  in  a  beaker,  allowing  to  settle,  and  pouring  the  liquid 
through  a  paper  filter.  The  filtrate  tested  with  the  iodine  solution  does 
not  give  a  blue  color.  Use  the  potato  starch  of  the  experiment  for  this 
test. 

When  boiled  with  dilute  acids  starch  is  converted  into  sol- 
uble compounds.  The  nature  of  these  compounds  depends 
on  the  acid  used  and  on  the  duration  of  the  heating.  By  pro- 
longed heating  glucose  is  the  main  product  of  the  reaction,  as 
already  illustrated,  but  various  intermediate  steps  may  be  rec- 
ognized, maltose  and  forms  of  dextrin  being  readily  demon- 
strated. With  strong  acids  the  results  are  quite  different. 
With  sulphuric  acid  the  reaction  is  completely  destructive, 
water,  carbon  dioxide  and  sulphurous  acid  from  reduction 
being  formed.  Strong  nitric  acid  acts  as  an  oxidizing  agent 
and  by  proper  manipulation  oxalic  acid  may  be  obtained  in 
quantity  as  a  product  of  the  oxidation. 

Ex.  Add  15  cc.  of  strong  sulphuric  acid  to  a  gram  of  starch  in  a  flask 
holding  about  200  cc.  Heat  to  the  boiling  point  and  observe  that  a  black 
mass  is  soon  produced.  By  prolonged  heating  this  is  further  decomposed, 
while  fumes  of  sulphurous  oxide  escape,  leaving  finally  a  colorless  liquid. 

Ex.  Add  15  cc.  of  strong  nitric  acid  to  one  gram  of  starch  in  a  flask 
holding  200  to  300  cc,  place  this  on  a  sand-bath  in  a  fume  chamber  and 
apply  heat.  After  a  time  copious  red  fumes  are  given  off.  Remove  the 
lamp  and  allows  the  reaction  to  continue  until  the  fumes  cease  to  be  evolved. 
Finally,  transfer  the  liquid  to  a  porcelain  dish  and  evaporate  to  a  small 
volume.  On  cooling,  a  crystalline  residue  remains  which  consists  mainly 
of  oxalic  acid. 

When  carefully  heated  starch  may  be  largely  converted  into 
a  form  of  dextrin,  which,  as  will  be  fully  explained  later,  is 
one  of  the  important  stages  in  the  common  transformations  of 


CARBOHYDRATES    AND    RELATED    BODIES.  45 

Starch.  The  reaction  is  employed  on  the  large  scale  in  the 
manufacture  of  British  gum  which  is  used  in  the  preparation 
of  size  and  paste  for  various  purposes. 

Ex.  Heat  about  lo  gm.  of  starch  in  a  porcelain  dish  on  a  sand-bath  to 
a  temperature  short  of  the  point  where  it  begins  to  scorch.  It  is  neces- 
sary to  stir  well  all  the  time,  and  continue  the  heat  ten  minutes  after  the 
starch  has  become  uniformly  yellowish  browti.  Then  allow  the  dish  to 
cool,  add  water  and  boil  thoroughly,  which  brings  part  of  the  product  into 
solution.  When  sufficiently  diluted  this  solution  can  be  filtered.  The  fil- 
trate is  precipitated  by  alcohol.  The  addition  of  a  few  drops  of  iodine 
solution  to  the  aqueous  liquid  gives  rise  to  a  reddish  color  characteristic 
of  dextrin. 

The  chief  uses  of  starch  have  been  referred  to  in  other  con- 
nections. Much  is  directly  employed  as  food  and  large  quantities 
are  converted  into-  glucose  as  shown  above.  The  production 
of  various  kinds  of  dextrin  and  British  gum  is  also  extremely 
important  and  consumes  enormous  quantities  of  starch.  In 
the  form  in  which  it  occurs  in  nature,  that  is  mixed  with 
other  substances  in  small  amount,  starch  is  the  most  abundant 
of  our  foodstuffs,  and  the  one  consumed  in  largest  amount. 
Great  interest  therefore  attaches  to  the  reactions  by  which 
this  starch  is  made  soluble  or  digested  as  a  step  in  its  assim- 
ilation. The  discussion  of  this  fundamental  point  will  be 
left,  however,  for  a  following  chapter,  when  the  theory  of 
digestive  operations  can  be  explained  as  a  whole. 

In  certain  plants  a  variety  of  starch  called  inulin  occurs.  It 
is  best  obtained  from  tubers  of  the  dahlia,  and  is  interesting 
from  the  fact  that  by  hydrolysis  it  yields  fructose  instead  of 
glucose.  It  differs  from  the  ordinary  starch  in  yielding  a 
true  solution  with  hot  water,  and  in  giving  a  yellow  instead 
of  a  blue  color  with  iodine. 

Glycogen,  or  animal  starch.  This  product,  which  is 
formed  in  the  liver,  is  related  in  many  ways,  both  chemically 
and  physiologically,  to  common  starch.  In  some  respects  it 
resembles  also  the  simple  sugars,  from  which  it  is  indeed 
•derived,  and  may  be  said  to  stand  between  them  and  vegetable 
starch.     It  is  readily  soluble  in  water,  giving,  however,  an 


46  PHYSIOLOGICAL    CHEMISTRY. 

opalescent  solution.  This  is  especially  characterized  by  a 
strong  action  on  polarized  light,  [a]^,  ^ -f  196°  to  213^, 
according  to  different  authorities.  Like  common  starch  gly- 
cogen is  a  reserve  material,  being  formed  from  the  absorbed 
sugar  of  the  digestive  process,  and,  in  turn,  being  reconverted 
into  sugar  from  the  liver  as  this  is  required  for  oxidation  in 
the  body.  After  death  the  store  of  glycogen  in  the  liver  rap- 
idly diminishes,  glucose  being  produced.  The  amount  of  gly- 
cogen present  in  the  liver  varies  greatly  with  the  diet  and  time 
after  eating.  It  may  make  up  12  to  16  per  cent  of  the  total 
weight  of  the  organ,  but  is  usually  much  below  this,  perhaps 
in  the  mean  2  to  3  per  cent.  In  addition  to  its  occurrence  in 
the  liver  glycogen  is  found  in  variable  amount  in  the  muscles, 
and  in  traces  in  other  body  tissues.  It  occurs  also  in  the  vege- 
table kingdom,  and  has  been  recogiiized  in  certain  fungi.  The 
laboratory  production  of  glycogen  and  some  of  the  simple 
reactions  are  illustrated  by  the  following  experiments,  while 
the  physiological  relations  will  be  reserved  for  further  discus- 
sion in  a  later  chapter. 

Ex.  Kill  a  rat  or  a  rabbit ;  remove  the  liver  as  quickly  as  possible,  and 
without  delay  cut  it  into  small  bits,  which  throw  into  a  vessel  of  boiling 
water.  The  weight  of  the  water  should  be  about  five  times  that  of  the 
minced  liver.  Boil  five  minutes,  then  remove  from  the  water  and  rub  up 
in  a  mortar  with  fine  clean  quartz  sand.  In  this  way  the  fragments  of 
liver  become  thoroughly  disintegrated.  The  contents  of  the  mortar,  sand 
as  well  as  liver,  are  thrown  into  boiling  water  again  and  kept  at  100°  15 
minutes.  At  the  end  of  this  time  enough  dilute  acetic  acid  must  be  added 
to  impart  a  faint  acid  reaction.  This  coagulates  and  precipitates  some 
albuminous  matters,  which  are  separated  when  the  hot  mixtlire  is  filtered. 
In  the  opalescent  filtrate,  which  must  be  collected  in  a  cold  beaker,  a  fur- 
ther precipitation  of  albuminous  matter  is  effected  by  adding  a  few  drops 
of  hydrochloric  acid  and  some  potassium  mercuric  iodide  as  long  as  a 
precipitate  forms. 

Filter  again  and  use  the  dilute  aqueous  solution  of  gb'cogen  resulting 
for  tests  below. 

Ex.  Evaporate  about  half  of  the  liquid  above  to  a  small  volume  and 
precipitate  impure  glycogen  as  an  amorphous  white  powder  by  addition  of 
strong  alcohol. 

Ex.  Add  a  little  tincture  of  iodine  to  a  small  portion  of  the  solution, 
and  note  the  red  color  produced.     This  color  is  discharged  by  heat.     Boil 


CARBOHYDRATES    AND    RELATED    BODIES.  4/ 

some  of  the  solution  with  dilute  hydrochloric  acid  ten  minutes ;  neutralize 
the  acid  nearly,  cool  and  again  test  with  iodine.  No  color  is  now  pro- 
duced, as  glycogen  has  disappeared  under  the  treatment,  having  been  con- 
verted into  sugar. 

It  has  been  remarked  above  that  after  death  the  store  of  glycogen  in 
the  liver  rapidly  disappears,  so  that  tests  applied  at  the  end  of  a  day  or 
two  fail  to  show  its  presence.     This  may  be  shown  as  follows : 

Ex.  Cut  some  common  beef  liver  from  the  market  into  small  bits  and 
extract  with  boiling  water.  Boil  longer  to  coagulate  protein  bodies,  after 
adding  some  sodium  sulphate.  Apply  the  iodine  test  for  glycogen,  which 
is  found  absent,  and  the  Fehling  test  for  sugar,  which  is  found  present  in 
quantity.  It  is  an  excellent  exercise  also  to  determine  the  amount  of  sugar 
which  may  be  obtained  from  a  given  weight  of  liver.  In  this  test  the 
extraction  must  be  repeated  with  several  portions  of  water. 

The  Gums.  Some  of  these  occur  in  nature  as  products 
related  to  the  pentose  group  of  sugars  referred  to  some  pages 
back.  Others  are  related  to  starch  and  on  transformation 
yield  finally  hexoses.  The  group  of  gums  includes  further 
the  dextrin  bodies  formed  from  starch  by  several  reactions, 
one  of  which  has  been  already  illustrated.  The  transforma- 
tion of  starch  by  the  action  of  weak  acids  or  enzymes  is  far 
from  being  a  simple  process  and  much  uncertainty  still  exists 
as  to  the  number  of  intermediate  products  between  the  parent 
substance  and  the  final  maltose  or  glucose.  Some  writers 
have  attempted  to  distinguish  several  well  defined  stages  in 
the  reaction  and  describe  as  definite  bodies  erythrodextrin, 
achroodextrin,  amylodextrin  and  maltodextrin.  The  first 
gives  a  violet-red  color  with  iodine  and  is  easily  precipitated 
by  alcohol;  the  second  gives  no  color  with  iodine,  but  is 
still  precipitated  by  alcohol.  It  shows  reducing  power  with 
Fehling's  solution,  and  may  be  looked  upon  as  one  of  the  end 
products  of  the  action  of  diastase  on  starch,  maltose  being  the 
other.  The  name  amylodextrin  is  given  to  a  product  of  dias- 
tatic  action  and  also  to  a  dextrin  formed  by  the  treatment  of 
starch  with  very  dilute  acids.  It  is  said  to  show  a  purple 
color  with  iodine,  and  to  exhibit  very  strong  dextro  rotation. 
The  existence  of  malto-dextrin  is  affirmed  by  several  writers, 
but  the  properties  of  the  substance  are  not  well  established. 


48  PHYSIOLOGICAL    CHEMISTRY. 

Some  authors  have  gone  so  far  as  to  recognize  several  modi- 
fications of  achroodextrin,  which  are  described  as  a,  /?  and  y 
forms,  and  which  differ  in  optical  properties  and  reducing 
power.  The  more  recent  extended  investigations  seem  to  dis- 
prove this  notion,  however,  and  the  most  that  can  be  safely 
said  is  that  along  with  maltose  an  end  product  is  produced  by 
diastatic  conversion  of  starch  which  is  probably  a  single  sub- 
stance. A\'hat  is  called  erythrodextrin  is  more  likely  to  be  a 
mixture,  possibly,  of  soluble  starch  and  achroodextrin.  Under 
the  name  crythrogranulose  a  similar  complex  has  been  de- 
scribed. In  a  later  chapter  on  the  action  of  ferments  more 
will  be  said  about  the  theory  of  the  transformation  of  starch 
into  these  products. 

The  true  dextrins  are  not  directly  fermentable  with  yeast, 
but  they  appear  to  be  aldehyde  bodies  and  as  such  have 
reducing  power.  They  react  also  with  phenyl  hydrazine  and 
yield  osazones,  which,  however,  are  not  easily  purified,  because 
of  their  solubility.  The  dextrins  have  a  slightly  sweetish  taste 
and  all  show  a  specific  rotation  about  [a],,  =  +  196.  Be- 
yond the  empirical  formula  CeHjoOg  it  is  not  possible  to  go  in 
describing  the  constitution  of  these  bodies. 

The  natural  vegetable  gums  are  often  mixtures  of  several 
substances,  and  but  few  of  them  have  been  studied.  Gum 
arable  and  gum  Senegal  are  the  potassium  and  calcium  salts  of 
arable  acid  to  which  the  formula  (C6Hio05)2  +  HgO  is  given. 
On  treatment  with  weak  sulphuric  acid  both  arabinose  and 
galactose  appear  to  be  formed.  Agar-agar  is  said  to  yield  lac- 
tose and  then  galactose,  while  cherry  gum  yields  arabinose. 

Cellulose.  The  cell  walls  of  vegetable  substances  consist 
of  cellulose  mixed  always  with  related  compounds  of  which 
a  body  called  lignin  is  the  most  important.  The  cellulose 
resists  the  action  of  strong  oxidizing  or  other  agents  much 
more  perfectly  than  do  the  accompanying  bodies,  and  may 
therefore  be  freed  from  them  by  various  treatments.  In 
washed  Swedish  filter  paper  we  have  an  illustration  of  nearly 


CARBOHYDRATES    AND    RELATED    BODIES.  49 

pure  cellulose,  as  all  the  other  bodies  in  the  original  fibers  have 
been  removed  by  the  bleaching  and  washing  processes  to  which 
the  raw  material  was  subjected  in  the  manufacture  of  the 
paper.  A  pure  cellulose  paper  may  be  made  from  wood  also, 
but  only  by  more  complicated  operations. 

The  pure  cellulose  is  characterized  by  insolubility  in  water, 
weak  acids,  alkalies,  alcohol  or  ether.  It  may  be  dissolved 
rather  readily  in  a  solution  known  as  Schweitzer's  reagent 
and  by  prolonged  digestion  w4th  acids  is  converted  into  hexoses 
and  pentoses. 

The  natural  celluloses  may  be  divided  roughly  into  three 
groups :  (a)  those  which  resist  hydrolytic  action  very  per- 
fectly and  are  not  capable  of  serving  as  foodstuffs  for  any 
animals ;  in  this  group  we  have  linen  and  cotton  fibers,  hemp, 
China  grass,  etc.  (&)  Those  which  are  less  resistant  to  hydro- 
lytic action  and  which  contain  active  CO  groups.  These 
bodies  may  be  called  oxycelluloses ;  they  yield  also  furfuralde- 
hyde  by  distillation  with  hydrochloric  acid.  In  this  group 
we  have  the  mass  of  the  material  found  in  the  fundamental 
tissues  of  flowering  plants,  and  a  large  part  of  ordinary  woody 
tissue.  This  lignified  tissue  is  made  up  of  compound  celluloses 
or  lignocelluloses  from  which  the  cellulose  proper  may  be 
isolated  in  a  variety  of  ways.  Some  of  the  bodies  in  this 
group  are  partly  digestible  and  have  some  value  as  foods  for 
the  herbivora.  (c)  In  this  third  group  we  have  substances 
described  as  pseudocelluloses  or  hemicelluloses  and  which  offer 
little  resistance  to  hydrolysis.  They  are  easily  attacked  by 
weak  acids  and  alkalies  and  also  suffer  digestion  by  enzymes, 
so  that  they  may  be  classed  among  the  foodstuffs  of  limited 
value  for  the  herbivora.  These  bodies  resemble  starch  much 
more  than  they  resemble  the  lignocelluloses.  By  action  of 
weak  acids  fermentable  sugars  are  formed  almost  quantita- 
tively from  pseudocelluloses,  while  from  the  lignocelluloses 
not  over  about  20  per  cent  of  fermentable  sugars  may  be 
obtained. 


50  PHYSIOLOGICAL    CHEMISTRY. 

Ex.  Prepare  Schweitzer's  reagent  by  first  precipitating  copper  hydrox- 
ide from  copper  sulphate  solution,  in  the  presence  of  a  little  ammonium 
chloride,  by  addition  of  sodium  hydroxide  in  excess.  Wash  the  precipi- 
tate thoroughly  and  then  dissolve  it  in  the  smallest  possible  quantity  of 
strong  ammonia  water.  This  yields  a  deep  blue  solution,  the  reagent  in 
question.  It  dissolves  cotton  rather  easily.  This  solution  may  be  filtered 
after  dihition,  and  from  the  filtrate  a  pure  cellulose  is  thrown  down  by 
addition  of  acids. 

By  action  of  strong  nitric  acid,  aided  by  sulphuric  acid,  cellulose  is  con- 
verted into  a  series  of  nitrates  or  "  nitro-celluloses."  The  number  of  Nd 
groups  added  depends  on  the  strength  of  the  acid  mixture  and  time  of  its 
action.  The  more  highly  nitrated  products  constitute  the  well-known  ex- 
plosives. Products  not  so  highly  nitrated  are  used  in  the  preparation  of 
collodion  and  celluloid.  This  latter  is  essentially  a  mixture  of  camphor 
and  nitrated  cellulose  from  cotton  or  paper.  These  bodies  are  esters  and 
therefore  may  be  decomposed  by  alkalies. 


CHAPTER    IV. 
THE    FATS    AND    SUBSTANCES    RELATED    TO    THEM. 

In  nature  we  find  a  large  number  of  esters  composed  of 
the  fatty  acids  united  to  glyceryl.  These  are  the  ordinary 
fats  and  as  foodstuffs  they  are  nearly  as  important  as  the 
carbohydrates.  In  structure  they  are  practically  all  of  the  type 
C3H5(C„H2„_i02)3,  but  include  bodies  of  widely  different 
physical  properties.  Some  are  liquids,  while  others  at  the 
ordinary  temperature  are  hard  solids.  Nearly  all  vegetable 
products  contain  fats  of  some  kind;  often  the  amount  is  very 
small,  but  frequently  it  constitutes  fully  50  per  cent  by  weight 
of  the  seed,  nut  or  fruit  in  question.  In  the  animal  kingdom 
fats  are  always  present,  in  some  amount,  in  all  organisms. 
The  animal  fats  are  often  derived  from  the  vegetable  fats  con- 
sumed as  food. 

THE    NATURAL    FATS. 

The  important  fatty  acids  combined  with  the  radical  of 
glycerol,  C3H5(OH)3,  are  given  in  the  following  table.  The 
combinations  are  essentially  like  that  illustrated  by  this  struc- 
tural formula  of  stearin : 

CH2  — O  — C1SH35O 

I 
CH  —  O  — C18H35O 

I 
CH2  — O  — C18H35O 

SATURATED   ACIDS,   CH^nO^, 

Formic  acid,  HCHO2         \ 

Acetic  acid,  HC2H3O2         [  glycerides  not  natural  substances. 

Propionic  acid,  HC3H5O2  -' 

Butyric  acid,  HC4H7O2,  occurs  in  butter  fat  as  glyceride. 

Pentoic  acid,  HC5H9O2,  valeric  acid  occurs  as  a  natural  compound. 

Caproic  acid,  HC6H11O2,  in  butter  fat  as  glyceride. 

CEnanthylic  acid,  HC7H13O2,  does  not  occur  as  glyceride. 

51 


52  PHYSIOLOGICAL    CHEMISTRY. 

Caprylic  acid,  HCsHuOj,  as  gljceride  in  butter  and  other  fats. 

Pelargonic  acid,  HC9H,702,  in  vegetable  kingdom,  but  not  as  glyceride. 

Capric  acid,  HC10H18O2,  in  butter  and  other  fats  as  glyceride. 

Undecylic  acid,  HCnHsiOi,  not  found  as  natural  glyceride. 

Laurie  acid,  HC12H23O2,  as  glycerol  ester  in  several  fats. 

Myristic  acid,  HCuHjtO-,  in  nutmeg  butter  and  other  fats  as  glyceride. 

Palmitic  acid,  HC16H31O2,  as  glyceride  in  many  fats. 

Margaric  acid,  HC1-H33O2,  obtained  as  artificial  glyceride. 

Stearic  acid,  HC1SH35O2,  as  glyceride  in  many  fats. 

Arachidic  acid,  HC20H38O2,  as  glyceride  in  peanut  oil. 

Behenic  acid,  HC22H4302,  as  glyceride  in  certain  oils. 

A  few  other  acids  of  this  series  are  known  in  glycerol  com- 
binations but  they  are  unimportant. 

NON-SATURATED  ACIDS,  C„H,,.-20,  AND  CMin-.O^- 

Not  many  of  these  acids  occur  as  natural  glycerides. 

Hypogaeic  acid,  C18H30O2,  as  glyceride  in  peanut  oil. 
Oleic  acid,  C18H34O2,  as  glyceride  in  many  oils. 
Linoleic  acid,  C18H32O2,  as  glyceride  in  drying  oils. 
Ricinoleic  acid,  C1SH34O3,  as  glyceride  in  castor  oil. 

A  large  number  of  these  acids  occur  in  the  edible  fats, 
while  some  are  found  in  products  of  little  importance,  or  in 
such  as  have  technical  uses  only.  The  fats  which  are  most 
commonly  used  as  foods  are  those  consisting  largely  or  wholly 
of  stearin,  palmitin  and  olein.  Butter  fat,  however,  contains 
relatively  large  amounts  of  other  glycerides. 

Reactions  of  Fats.  Certain  general  reactions  are  common 
to  practically  all  fats  and  will  be  explained  here  in  detail. 

Saponification.  This  term  is  applied  to  the  change 
which  follows  when  fats  are  treated  with  alkali  solutions, 
usually  with  application  of  heat.  The  fats  decompose  more 
or  less  readily  and  as  products  we  have  soaps  and  glycerol, 
according  to  these  equations  : 

C3H5(CisH3502)3  +  3KOH  =  CaHsCOH),  +  3KC18H36O. 

2C3H=(GsH3302)3  +  3PbO  +  3H2O  =  2C3H5(OH)3  +  3Pb(G8H3302)2 

In  the  first  case  potassium  stearate  is  formed  and  in  the 
second  lead  oleate.  which  is  the  important  constituent  of  lead 


FATS    AND    RELATED    SUBSTANCES.  53 

plaster.  Here  lead  oxide  and  water  are  equivalent  to  lead 
hydroxide.  Much  of  the  glycerol  of  commerce  is  produced 
by  such  decompositions.  When  sodium  hydroxide  is  used 
with  the  common  fats  ordinary  hard  soap  results. 

An  analogous  change  occurs  when  fats  are  subjected  to  the 
action  of  water  at  a  high  temperature  or  superheated  steam. 
We  have  here  hydrolysis  purely,  although  the  term  saponifica- 
tion by  steam  is  sometimes  applied.  The  same  reaction  is 
brought  about  by  certain  enzymes  at  the  ordinary  tempera- 
ture, for  example  by  the  enzyme  known  as  lipase  in  the  pan- 
creatic secretion.  This  will  be  discussed  fully  later  as  it  is 
important  in  the  digestion  of  fats.  Sometimes  the  reaction 
is  complete  as  shown  by  this  equation : 

C3H5(CisH3502)3  +  3H.O  =  C3H5(OH)3  +  sCisHa.O. 

But  products  of  partial  hydrolysis,  monostearin  and  distearin, 
for  example,  may  be  left,  as : 

f  C18H35O2  r  OH 

C3H5  j  GSH35O2  -f-  H2O  =  GH=  j  C18H35O2  +  HC1SH35O2 
l  C1SH35O2  (  C1SH3BO3 

ILLUSTRATIVE    TESTS. 

Some  of  the  saponifications  are  illustrated  by  these  experi- 
ments : 

Ex.  Boil  about  25  gm.  of  tallow  with  a  solution  of  10  gm.  of  potas- 
sium hydroxide  in  100  cc.  of  water.  Stir  the  mixture  thoroughly  until 
it  becomes  homogeneous ;  that  is,  until  no  oil  globules  are  seen  floating 
on  top  of  the  aqueous  liquid,  which  may  require  half  an  hour.  Add  water 
from  time  to  time,  to  make  up  for  that  lost  by  evaporation.  The  result- 
ing mass  is  a  mixture  of  glycerol,  excess  of  alkali  and  soft  soap.  To  this 
now  add  a  solution  of  15  gm.  of  common  salt  in  75  cc.  of  water  and  heat 
again,  which  brings  about  a  conversion  of  the  soft  soap  into  the  hard  or 
sodium  soap.  On  cooling  this  separates  and  floats  on  the  excess  of  spent 
lye  and  salt  solution. 

Ex.  The  presence  of  fatty  acids  in  the  above  soap  can  be  shown  by 
adding  to  a  portion  of  it  enough  hydrochloric  acid  to  decompose  the  soap. 
Use  about  half  the  product  of  the  experiment,  dilute  with  water,  and  add 
the  acid  in  slight  excess,  about  10  cc.  of  the  strong  commercial  acid. 
Warm  on  the  water-bath,  which  will  cause  the  liberated  fatty  acids  to  col- 
lect on  the  surface  as  a  liquid  layer  as  soon  as  the  temperature  becomes 


54  PHYSIOLOGICAL    CHEMISTRY. 

high  enough.  Add  more  water  and  allow  the  whole  to  cool.  A  semi- 
solid layer  of  fatty  acids  can  now  be  lifted  from  the  surface  of  the  liquid. 
The  hardness  of  the  mixed  acids  depends  on  the  fat  taken  for  experiment. 
Mutton  and  beef  tallows  yield  very  solid  acids ;  with  lard  the  mass  is 
softer,  while  with  some  oils  the  acids  do  not  solidify  at  all  at  the  ordi- 
nar}'^  temperature. 

Ex.  Dissolve  a  small  portion  of  the  fatty  acids  in  warm  alcohol,  nearly 
to  saturation.     On  cooling,  the  acids  separate  in  crystalline  scales. 

Ex.  The  presence  of  glycerol  as  one  of  the  products  formed  by  the 
saponification  of  fats  is  best  shown  as  follows : 

Mix  50  cc.  of  cottonseed  oil  with  25  gm.  of  litharge  and  100  cc.  of 
water  in  a  porcelain  dish.  Place  over  a  Bunsen  burner  on  gauze  and  stir 
until  all  oil  globules  have  disappeared,  adding  a  little  water  from  time  to 
time.  The  litharge  with  water  acts  as  lead  hydroxide  and  saponifies  the 
fat,  forming  an  insoluble  lead  soap,  or  plaster,  and  glycerol.  When  the 
saponification  is  complete  add  more  water,  heat  and  stir  well  to  dissolve 
gh'cerol.  Allow  to  settle  a  short  time  and  pour  the  aqueous  solution 
through  a  filter.  To  the  residue  add  water  again,  heat,  allow  to  settle 
and  pour  through  the  same  filter.  Concentrate  the  mixed  filtrates  to  a 
small  volume  and  after  cooling  observe  the  sweet  taMe  of  the  thickish 
residue. 

Ex.  Dissolve  a  portion  of  the  sodium  soap  in  water  with  aid  of  a  little 
alcohol.  Then  add  some  solution  of  calcium  chloride  or  lead  acetate.  A 
white  precipitate  is  formed  as  the  calcium  and  lead  salts  of  the  fatty  acid 
are  not  soluble  in  water.  Hard  waters,  which  contain  salts  of  calcium 
and  magnesium,  decompose  soap  in  the  same  manner. 

Other  Reactions.  The  common  fats  are  insoluble  in  water 
and  when  mixed  with  the  latter  tend  to  separate  immediately. 
However,  it  is  possible  to  convert  the  fats  and  water  into  a 
peculiar  mixture  called  an  emulsion  which  does  not  separate 
into  two  layers  on  standing.  In  this  condition  the  fat  consists 
of  extremely  minute  globules  which  remain  in  suspension  and 
which  may  be  passed  through  the  pores  of  coarse  filter  paper. 
It  does  not  seem  possible  to  secure  an  emulsion  with  perfectly 
neutral  fats,  and  in  most  cases  the  phenomenon  depends  on  the 
presence  of  a  trace  of  soap  formed.  In  the  processes  of  diges- 
tion of  fats  in  the  animal  body  emulsification  plays  a  very 
important  part  as  will  be  shown  later.  It  follows  probably 
the  partial  hydrolysis  of  the  fats  by  lipase,  referred  to  above. 

As  they  exist  in  the  animal  or  vegetable  organism  the  fats 
are  doubtless  all  amorphous  substances,  but  in  the  separated 
condition  the  solid  fats  always  become  more  or  less  crystalline. 


FATS  AND  RELATED  SUBSTANCES.  55 

This  may  be  readily  shown  by  dissolving  some  fat  in  a  proper 
solvent  which  is  then  allowed  to  evaporate  slowly. 

The  common  fats  can  not  be  distilled  under  the  ordinary 
pressure  without   decomposition,   and   the   distillation   of  the 


Fig.    4.     Mutton    tallow    crystal-         Fig.    5.     Cat    fat    crystallized    from 
lized    from    chloroform.     300    diam-  chloroform.     300    diameters. 


eters. 


Fig.     6.       Beef     tallow     crystal-  Fig.     7.       Beef     tallow     crystal- 

lized   from    chloroform.     300    diam-         lized    from    chloroform.     300    diam- 
eters, eteirs. 

fatty  acids  is  also  difficult.  When  the  common  fats  are 
strongly  heated  they  emit  a  peculiar  odor,  due  to  the  acrolein 
formed  by  the  partial  destruction  of  glycerol.  The  following 
experiments  illustrate  some  of  the  points  referred  to : 


56  PHYSIOLOGICAL    CHEMISTRY. 

Ex.  Note  the  solubility  of  small  bits  of  tallow  in  ether,  chloroform, 
benzine  and  alcohol,  using  in  each  case  the  same  volume  of  liquid,  with 
equal  weights  of  fat.  The  solubility  in  alcohol  will  be  found  much  less 
than  in  the  other  menstrua. 

Ex.  Dissolve  some  mutton  or  beef  tallow  in  chloroform  and  with  a 
glass  rod  put  two  or  three  drops  of  the  nearly  saturated  solution  on  the 
center  of  a  glass  slide.  As  the  chloroform  evaporates  a  film  begins  to 
form  on  the  top  of  the  drop.  Now  put  on  a  perfectly  clean  cover  glass 
and  allow  to  stand  until  crystallization  is  complete,  wliich  may  require  only 
a  few  minutes  or  some  hours,  the  time  necessary  depending  on  the  tem- 
perature and  on  the  concentration  of  the  solution.  Examine  the  crystals 
with  a  microscope.  Use  a  power  of  200  to  300  diameters.  By  repeating 
the  experiment  with  different  fats  considerable  variation  in  the  form  of 
the  crj-stals  may  be  noticed,  which  is  shown  in  the  annexed  cuts. 

Ex.  Add  to  5  cc.  of  cottonseed  oil  half  its  volume  of  strong  white  of 
egg  solution  and  shake  thoroughly.  The  liquids  mix  and  form  a  white 
mass  or  emulsion  which,  however,  is  not  usually  stable. 

Ex.  To  5  cc.  of  cottonseed  oil  containing  a  little  free  fatty  acid  add  10 
drops  of  strong  sodium  carbonate  solution  and  shake.  A  good  stable 
emulsion  is  made  in  this  way,  as  the  sodium  of  the  alkali  solution  forms 
a  soap  with  the  free  acid  and  this  appears  to  form  a  film  around  the  little 
fat  globules  which  prevents  their  flowing  together  again. 

Origin  of  Fats  in  the  Body.  The  question  of  the  forma- 
tion of  fats  in  the  animal  organism  has  been  much  discussed. 
It  was  once  assumed  that  Hke  protein  substances  the  fats  are 
products  of  the  vegetable  world  only,  and  that  the  animal  has 
not  the  power  of  building  them  up  from  compounds  which 
are  not  fats.  But  this  view  is  not  correct  as  we  have  abun- 
dant proof  that  fats  may  be  made  in  other  ways.  Much  has 
been  learned  from  the  results  of  cattle  feeding  experiments 
carried  out  in  agricultural  experiment  stations,  where  the  gain 
in  fat  is  often  much  in  excess  of  what  could  be  accounted  for 
by  the  amount  of  fats  in  the  food  consumed.  This  gain  must 
in  some  way  be  due  to  the  effect  of  the  carbohydrate  and  protein 
substances  in  the  rations  fed.  The  fattening  power  of  sugar 
has  long  been  recognized,  but  this  has  been  in  part  accounted 
for  on  the  theory  that  the  sugar  acts  to  protect  the  fats  of  the 
body  from  oxidation,  by  being  readily  oxidized  itself  to  keep 
up  the  body  energy.  But  much  evidence  has  been  accumu- 
lated to  show  that  carbohydrates  take  part  directly  in  the  pro- 
duction of  fats.     How  this  is  accomplished  is  not  known,  but 


FATS    AND    RELATED    SUBSTANCES.  57 

in  the  processes  syntheses  and  oxidations  both  must  be  con- 
cerned since  the  fat  molecules  are  more  complex  than  those  of 
the  carbohydrates. 

It  is  further  likely  that  protein  compounds  are  important 
factors  in  fat  production.  Many  writers  have  indeed  assumed 
that  we  have  in  the  breaking  down  of  protein  molecules  the 
chief  sources  of  fats,  but  this  view  has  been  strongly  com- 
bated. Indirectly  the  transformation  may  follow  in  this  way : 
It  "is  known  that  sugars  are  formed  as  cleavage  products 
of  albumins  in  the  ordinary  katabolic  processes  of  the  body 
and  possibly  a  portion  of  the  sugars  thus  formed  may  be  then 
built  up  to  produce  fats. 

The  existence  of  the  substance  known  as  adipocere  has 
long  been  held  to  furnish  a  pretty  strong  proof  of  the  produc- 
tion of  fatty  acids  from  protein.  This  adipocere  or  cadaver 
wax  is  often  found  in  large  masses  in  old  cemeteries  and 
consists  of  fatty  acids,  calcium  and  ammonium  soaps  in  the 
main.  It  is  held  that  this  substance  could  not  possibly  have 
come  from  the  small  amounts  of  fat  ordinarily  present  in 
cadavers  but  must  have  been  produced  from  the  muscular 
portions  of  the  body.  This  view  has  met  with  objections, 
however,  and  attempts  are  made  to  account  for  the  develop- 
ment of  the  adipocere  in  other  ways. 

In  the  body  fats  constitute  a  reserve  material  in  which  poten- 
tial energy  is  conveniently  stored  up.  In  sickness  or  in  wast- 
ing diseases  where  there  is  a  partial  or  complete  failure  in 
nutrition  this  fat  is  called  upon  to  supply  the  needs  of  the 
body.     The   fats   are  oxidized  while  the  muscular  tissue  is 

preserved. 

PHYSIOLOGICALLY  IMPORTANT  FATS. 

Stearin  or  Tristearin,  C3H5( €13113502)3.  This  is  a 
simple  fat  which  does  not  occur  in  nature  unmixed  with 
other  fats.  When  separated  in  purest  possible  condition  it 
shows  a  melting  point  of  55°  to  58°.  It  is  the  hardest  of  the 
common  simple  fats  and  apparently  the  least  soluble  in  alcohol 
or  ether.     It  may  be  separated  in  the  form  of  rectangular 


58  PHYSIOLOGICAL    CHEMISTRY. 

plates  by  crystallizing  from  hot  alcohol.  Stearic  acid  may  be 
obtained  in  the  form  of  pearly  crystalline  plates  or  scales.  It 
melts  at  71°. 

Palmitix  or  Tripalmitix,  C3H5(CicH3i02)3.  The  per- 
fect separation  of  this  fat  from  stearin,  with  which  it  is  usually 
associated  is  a  matter  of  considerable  difficulty.  The  fats  are 
much  alike.  The  melting  point  is  variously  stated  by  different 
observers,  but  appears  to  be  about  51°.  A  mixture  of  stearin 
and  palmitin  was  formerly  supposed  to  be  a  distinct  fat  and 
was  called  margarin,  C3H5(Ci7H3302)3.  This  fat  has  been 
produced  artificially  but  it  does  not  appear  to  be  a  natural 
product.  Palmitic  acid  resembles  stearic  acid  in  appearance 
and  solubility;  both  acids  are  slowly  soluble  in  strong  hot 
alcohol  and  yield  crj-stalline  plates  on  cooling.  The  melting 
point  of  palmitic  acid  is  about  62°. 

Olein  or  Triolein,  C3H5(Ci8H3302)3.  This  is  a  liquid 
fat  at  the  ordinary  temperature  and  is  a  constituent  of  most 
of  the  natural  fats  and  oils.  Some  fatty  oils  are  nearly  pure 
olein  and  become  solid  at  a  low  temperature.  The  soft  con- 
sistence of  lard,  human  fat  and  several  other  natural  mixtures 
is  due  to  the  olein  present.  Olein  is  a  nonsaturated  fat  and 
will  therefore  show  an  absorption  coefficient  as  explained 
below.  By  the  action  of  reagents  yielding  nitrous  acid  it  is 
converted  into  an  isomeric  substance  known  as  elaidin. 

Oleic  acid  in  pure  condition  is  not  very  stable,  because  of 
its  unsaturated  structure.  It  is  an  oily  liquid  at  the  ordinary 
temperature,  but  below  14"  is  converted  into  a  crystalline 
solid.  By  treatment  with  nitrous  acid  it  yields  the  isomeric 
elaidic  acid.  Oleic  acid  is  characterized  by  forming  a  lead 
salt  which  is  soluble  in  ether  while  lead  palmitate  and  stearate 
are  practically  insoluble. 

Lard  and  Tallow  are  essentially  mixtures  of  the  three 
fats,  palmitin,  stearin  and  olein.  By  heating  lard  to  its  melt- 
ing point,  cooling  slowly  and  subjecting  the  warm  mass  to 
pressure  in  a  filter  press  the  softer  portion,  consisting  mainly 
of  olein,  may  be  separated.     This  is  known  as  lard  oil,  while 


FATS  AND  RELATED  SUBSTANCES.  59 

the  harder  residue  is  sometimes  caUed  lard  stearin.  It  has 
about  the  consistence  of  butter.  By  subjecting  beef  suet  to 
the  same  treatment  a  soft  portion  known  as  olco  oil  is  sepa- 
rated from  a  solid  residue  called  beef  stearin.  The  oleo  oil 
is  the  material  most  often  employed  under  the  name  of  oleo- 
margarin  as  a  substitute  for  butter.  A  mixture  of  somewhat 
similar  consistence  is  made  in  other  ways ;  for  example  by 
combining  cottonseed  oil  with  beef  stearin. 

Oleomargarin  is  the  name  given  by  law  to  these  butter 
substitutes  in  the  United  States.  Sometimes  the  fats  are 
churned  with  milk  or  mixed  with  a  certain  amount  of  real 
butter  to  furnish  a  product  with  flavor  suggesting  butter. 
The  name  butterine  is  usually  given  to  such  mixtures  and 
when  properly  made  they  are  wholesome  and  in  every  way 
as  good  as  butter  from  the  standpoint  of  nutritive  value. 

Butter.  The  fat  of  milk  is  a  very  complex  mixture  and 
an  exact  separation  has  not  yet  been  made  by  the  methods  of 
chemical  analysis.  According  to  the  older  notion  butter  fat 
contains  essentially  olein,  stearin  and  palmitin,  with  a  little 
butyrin,  to  which  the  flavor  and  odor  are  largely  due,  but  it 
has  been  shown  that  other  glycerol  esters  are  certainly  present. 
The  results  of  some  recent  examinations  may  be  approximately 
expressed  as  follows : 

Glyceryl   butyrate    7.0 

Glyceryl  caproate,  caprylate  and  caprate 2.0 

Glyceryl    oleate    36.0 

Glyceryl  myristate,  palmitate  and  stearate 55.0 

lOO.O 

From  various  investigations  it  appears  that  these  fats  are 
not  present  as  simple  esters,  but  may  possibly  exist  in  combi- 
nations represented  by  formulas  like  this : 

r  GSH35O2 

CsHb    -{    C16H31O2 
{   C.Ht02 

The  melting  point  of  butter  fat  is  between  38°  and  45°. 
On  melting  100  parts  by  weight  of  pure  butter  fat,  saponifying, 


60  PHYSIOLOGICAL    CHEMISTRY. 

separating  the  fatty  acids  and  wasliing  out  eventliing  solu1~)le 
in  hot  water  (butyric  acid,  etc.)  it  is  found  that  the  insoluble 
residue  left  amounts  in  the  mean  to  88  per  cent,  but  may  be 
more  or  less  with  different  grades  of  butter.  Commercial 
butter  contains  in  the  mean  about  85  per  cent  of  fat,  10  per 
cent  of  water  and  5  per  cent  of  salt. 

Human  Fat.  This  consists  essentially  of  olein,  palmitin 
and  stearin.  In  the  fat  of  children  the  solid  glycerides  appar- 
ently are  in  excess,  while  in  later  life  the  proportion  of  olein 
increases,  so  that  the  separated  fat  may  appear  quite  soft.  In 
the  human  adult  the  olein  may  amount  to  75  per  cent  of  the 
whole  fat,  but  the  proportion  varies  with  different  parts  of 
the  body. 

Glycerol.  Since  it  is  a  constituent  of  all  the  true  fats  a 
few  words  about  this  alcohol  will  be  in  order  here.  The  sub- 
stance was  first  recognized  in  the  aqueous  liquid  left  in  the 
preparation  of  lead  plaster  and  for  many  years  all  used  in 
pharmacy  and  in  the  manufacture  of  cosmetics  was  made  by 
the  same  reactions.  Since  its  importance  in  technology  was 
recognized  it  has  been  produced  on  the  large  scale  by  other 
kinds  of  saponification  or  hydrolysis.  In  pure  condition 
it  is  a  thick,  sweetish  liquid  with  a  specific  gravity  of  1.266 
at  15°  referred  to  water  at  the  same  temperature.  It  mixes 
with  water  and  alcohol  in  all  proportions,  but  is  not  soluble 
in  pure  chloroform,  benzene,  carbon  disulphide  or  petroleum 
ether.  It  is  very  slightly  soluble  in  ether.  Like  other  alcohols 
it  may  be  combined  with  acids  to  form  esters.  With  stearic 
acid  mono-,  di-  and  tri-stearin  are  produced,  the  last  being 
identical  with  the  natural  fat.  When  fed  to  animals  glycerol 
may  be  oxidized  to  a  limited  extent  only.  Any  excess  of  it 
fails  to  be  assimilated  and  soon  produces  disorders  in  digestion 
and  absorption.  Its  food  value,  in  free  form,  is  therefore  very 
slight. 

Recognition  and  Determination  of  Fats.  In  physiological  chemical  in- 
vestigations fats  are  separated  from  accompanying  substances  through 
their  solubility  in  warm  ether,  chloroform  or  petroleum  ether.     The  carbo- 


FATS    AND    RELATED    SUBSTANCES.  6 1 

hydrates,  protein  bodies  and  salts  are  not  soluble  in  these  liquids.  The 
saponification  test  is  also  of  value  in  identification.  Many  of  the  fats  con- 
tain unsaturated  acid  groups  and  are  therefore  able  to  absorb  certain 
amounts  of  halogens  from  specially  prepared  solutions.  Oleic  acid, 
C1SH34O2,  absorbs  iodine  or  bromine  to  form  C18H34I2O2  or  CisH34Br202. 
The  fat  to  be  examined  is  dissolved  in  chloroform  and  treated  with  the 
standard  solution.  After  a  time  the  excess  of  iodine  (or  bromine)  is  de- 
termined and  that  absorbed  by  the  fat  is  a  measure  of  the  non-saturated 
acid  present.  Linoleic  acid  absorbs  twice  as  much  iodine  or  bromine  as 
oleic  acid,  as  the  formula  C1SH32O2  becomes  C1SH32I4O2. 

The  determination  of  the  amount  of  insoluble  fatty  acids  furnished  by 
a  given  weight  of  fat  is  also  a  valuable  factor  in  the  study  of  these  bodies. 
In  another  method  the  fat  is  saponified,  the  soap  formed  decomposed  by 
dilute  sulphuric  acid  and  the  resultant  product  subjected  to  distillation. 
Fatty  acids  which  are  volatile  pass  over  and  collect  in  the  distillate,  where 
their  amount  may  be  determined  in  terms  of  KOH  or  NaOH  by  titration. 
In  this  treatment  stearin  and  palmitin  yield  acids  not  volatile  with  steam. 
This  is  a  valuable  test  and  is  applied  in  the  examination  of  butter  sup- 
posed to  be  adulterated  with  other  fats. 

Lecithin.  This  is  a  peculiar  complex  body  which  con- 
tains phosphoric  acid  and  an  organic  basic  group  in  place  of 
one  of  the  fatty  acid  radicals  in  the  common  fats.  It  is 
found  in  the  vegetable  kingdom,  but  commonly  and  most  char- 
acteristically in  many  animal  tissues,  in  the  brain  and  nerve 
tissue,  blood,  lymph,  milk,  pus,  yolk  of  egg,  etc.  It  is  most 
readily  prepared  from  the  last  named  substance.  The  follow- 
ing formula  represents  the  supposed  structure  of  the  body. 

r  —  O  —  C1SH35O 

C3H3  -<  —  O  —  CisHsoO  f  / i^TT  \ 

I  —  O  —  PO  •  HO  •  O  •  C2H,  -  N  I  5pf 

in  which  distearylglycero-phosphoric  acid  is  combined  with 
the  base  choline: 

(CH3)3-n{OH^qj^ 

It  appears  that  several  forms  of  lecithin  exist,  containing 
oleic  and  palmitic  acid  as  well  as  stearic.  They  undergo 
saponification,  yielding  fatty  acids,  glycero-phosphoric  acid  and 
choline.  They  are  soluble  in  ether  and  alcohol  and  in  other 
respects  resemble  the  true  fats.  With  water  lecithin  swells 
to  a  gelatinous  mass  which  under  the  microscope  presents  a 


62  PHYSIOLOGICAL    CHEMISTRY. 

characteristic  appearance.  The  function  of  lecithin  in  the 
body  is  not  understood,  but  from  the  fact  of  its  wide  distri- 
bution and  its  occurrence  in  milk  it  is  reasonable  to  assume 
that  it  performs  some  important  part. 

The  Waxes.  These  bodies  bear  some  resemblance  to  the 
fats  and  will  be  briefly  mentioned  here.  They  consist' largely 
of  esters  of  the  higher  monohydric  alcohols  of  the  saturated 
series,  and  in  most  cases  are  complex  mixtures  of  which  the 
composition  is  not  exactly  known.  Spermaceti  seems  to  con- 
sist largely  of  cetyl  palmitate,  CioH,^;jOCi6H3iO.  Beeswax 
contains  some  free  acid,  cerotic  acid,  in  addition  to  the  esters. 
The  most  important  constituent  is  apparently  myricin  or 
myricyl  palmitate,  CooHdOCioHsjO.  The  waxes  are  not 
easily  saponified  and  as  a  rule  clear  soap  solutions  are  not 
obtained. 

Cholesterol.  This  substance  is  an  alcohol,  but  in  appear- 
ance it  resembles  some  of  the  solid  fats  and  is  associated  with 
them  in  several  natural  products.  Hence  it  is  in  place  to 
describe  it  here.  The  formula  C27H45OH  probably  repre- 
sents the  composition  of  the  body  which  is  found  in  the  brain, 
yolk  of  egg,  the  liver,  blood  and  other  tissues  and  is  especially 
abundant  in  the  fat  of  wool.  It  constitutes  also  the  main 
substance  in  certain  gall-stones  from  which  it  may  be  sepa- 
rated in  nearly  pure  condition.  In  wool  fat  it  exists  partly  in 
the  free  state  and  partly  in  combination  with  fatty  acids  in 
the  form  of  esters. 

It  is  readily  soluble  in  hot  alcohol,  ether,  benzene  and  chloro- 
form, but  not  in  water  or  alkali  solutions.  It  is  therefore  left 
as  an  insoluble  residue  in  the  saponification  of  fatty  mixtures 
containing  it.  Under  the  microscope  pure  cholesterol  appears 
as  a  mass  of  white  plates  with  sharp  angles.  The  cholesterol 
esters  combine  with  water  to  form  stable  emulsions,  and  it  is 
probably  on  account  of  the  presence  of  these  esters  that  the 
substance  known  as  lanolin  is  practically  valuable.  This  lano- 
lin is  made  from  purified  wool  fat  and  is  largely  used  in  the 
preparation  of  salves  and  ointments. 


FATS    AND    RELATED    SUBSTANCES.  63 

An  isomeric  substance  known  as  isocholesterol  is  often 
found  associated  with  the  true  cholesterol,  especially  in  wool 
fat.  In  the  vegetable  kingdom  other  forms  of  cholesterol 
are  widely  distributed  in  small  quantities,  being  found  in  most 
oils  and  seeds.  All  forms  of  cholesterol  have  a  marked  action 
on  polarized  light. 

Ex.  If  gall-stones  are  obtainable  the  following  reactions  may  be  carried 
out  in  illustration  of  properties  of  cholesterol.  Crush  the  stones  to  a 
powder  and  boil  with  water  to  remove  anything  soluble.  Extract  the 
residue  several  times  with  hot  alcohol,  filter,  unite  the  solutions  and  allow 
the  cholesterol  to  crystallize  on  cooling.  As  some  fat  may  be  present  this 
must  be  removed  by  saponifying  with  a  little  alcoholic  potassa  solution. 
After  saponification  boil  off  the  alcohol  and  extract  the  dry  residue  with 
ether,  in  which  soaps  are  insoluble.  On  evaporation  of  the  ether  a  nearly 
pure  cholesterol  is  obtained.  It  may  be  further  purified  by  recrystalliza- 
tion  from  hot  alcohol.     With  the  substance  these  tests  may  be  made : 

Salkowski's  Test.  Dissolve  about  10  milligrams  of  cholesterol  in  2 
cubic  centimeters  of  chloroform  and  shake  with  an  equal  volume  of  strong 
sulphuric  acid.  The  chloroform  becomes  colored  blood  red,  then  cherry 
red  and  finally  purple.  The  acid  shows  a  dark  green  fliuorescence.  If 
the  chloroform  is  poured  into  a  dish  the  color  changes  to  blue,  then  green 
and  finally  yellow. 

BuRCHARD-LiEBERMANN  Test.  Dissolve  about  10  milligrams  of  choles- 
terol in  2  cubic  centimeters  of  chloroform,  add  20  drops  of  acetic  anhy- 
dride and  I  drop  of  strong  sulphuric  acid.     A  violet  pink  color  results. 

The  appearance  of  cholesterol  plates  should  be  studied  under  the 
microscope. 

The  presence  of  cholesterol  in  the  ester  form  in  lanolin  and 
similar  preparations  of  wool  fat  may  be  shown  by  the  above 
tests. 


CHAPTER    V. 

THE    PROTEIN    SUBSTANCES. 

These  extremely  important  bodies,  usually  called  albumi- 
nous bodies,  are  found  in  vegetable  and  animal  organisms  of 
all  kinds  and  in  some  form  are  essential  elements  in  cell 
growth.  Unlike  the  fats  and  carbohydrates  they  seem  to  be 
elaborated  in  the  vegetable  kingdom  only ;  or,  at  any  rate,  the 
fundamental  structures  in  them  appear  to  be  fonned  in  vege- 
table growth  only.  The  animal  is  able  to  modify  and  trans- 
form to  some  extent,  but  apparently  can  not  build  them  up 
from  simple  materials.  In  composition  the  protein  bodies  are 
extremely  complex;  qualitatively  they  contain  carbon,  hydro- 
gen, oxygen,  nitrogen  and  sulphur.  In  an  important  group 
of  these  bodies  phosphorus  is  also  present  and  a  few  contain 
iron.  The  quantitative  composition  of  some  of  the  best  known 
protein  compounds  is  expressed  approximately  as  follows : 

Per  Cent. 

C  50.0-55.0 

•              H  6.5-  y.i 

O  19.0-23.0 

N  1 5.0-1 7.0 

S  0.3-  2.4 

Attempts  have  been  made  to  calculate  formulas  for  certain 
protein  bodies  from  the  results  of  analyses,  but  no  great  impor- 
tance attaches  to  the  empirical  formulas  so  reached.  The 
best  analyses  made  of  the  compounds  differ  among  themselves 
to  an  extent  that  makes  a  definite  result  quite  impossible. 
This  is  largely  due  to  the  fact  that  there  are  great  practical 
difficulties  in  the  way  of  properly  purifying  the  substances  as 
a  preliminary  to  analysis;  they  all  occur  mixed  with  other 
compounds,  such  as  fats,  carbohydrates  and  mineral  matters, 
and  to  remove  these  without  in  any  way  altering  the  compo- 

64 


THE    PROTEIN    SUBSTANCES.  65 

sition  of  the  complex  albuminous  molecule  is  extremely  diffi- 
cult, if  not  impossible.  The  formulas  which  have  been  pub- 
lished are  interesting  chiefly  in  showing  roughly  how  complex 
the  structures  certainly  are.  For  serum  albumin  Hofmeister 
has  given  this  minimum  formula : 

while  for  egg  albumin  he  has  given  this : 

C239H3S6N5SS2OTS. 
Even  more  complex  formulas  are  given,  for  example  this  : 

C755Ml215Nl95blo0235. 

These  formulas  are  in  a  measure  based  on  an  assumption  as 
to  the  number  of  sulphur  atoms  present,  about  which  some- 
thing will  be  said  later. 

The  usual  methods  of  fixing  organic  formulas  by  aid  of  a 
molecular  weight  determination  can  not  be  successfully  applied 
in  these  cases.  In  the  cryoscopic  method,  for  example,  the 
traces  of  mineral  impurities  present  have  possibly  as  much 
influence  on  the  result  as  the  whole  weight  of  dry  protein. 
Because  of  changes  in  composition  at  a  high  temperature  the 
boiling  point  method,  even  if  otherwise  reliable,  can  not  be 
applied,  and  methods  based  on  osmotic  pressure  observations 
lead  to  results  of  no  value. 

CLASSIFICATION    OF    THE    PROTEIN    BODIES. 

The  substances  thus  far  studied  have  been  divided  into 
groups  or  classes  dependent  on  chemical  composition  or  struc- 
ture. With  the  protein  compounds  this  is  only  partially 
possible  because  of  our  lack  of  full  knowledge  in  this  direction. 
Of  the  structural  relations  of  the  protein  molecules  nothing 
whatever  is  known,  while  of  composition  only  a  few  general 
facts  are  clearly  enough  established  to  be  available  in  a  scheme 
of  classification.  The  first  efforts  at  classification,  which 
we  owe  largely  to  the  work  of  Hoppe-Seyler,  were  therefore 
essentially  empirical.  Other  schemes  were  later  proposed  as 
6 


66 


PHYSIOLOGICAL    CHEMISTRY. 


more  facts  were  brouglit  to  lig-ht,  so  that  finally  a  grouping 
like  the  following  came  to  be  gradually  accepted  by  physio- 
logical chemists,  with  slight  differences  in  details  only.  The 
arrangement  below  is  that  of  Hammarsten,  in  the  form  given 
by  Cohnheim.     He  makes  four  principal  divisions  as  follows : 

True  or  Native  Albumins. 

Derived  Albumins  or  Transformation 

Products. 
Proteids. 
Albumoids. 


Protein  Bodies 


These   four   great    divisions    mav    be    further    subdivided : 


TRUE  OR  NATIVE 
ALBUMINS. 


DERIVED  OR 

TRANSFORMATION 

PRODUCTS. 


PROTEIDS. 


ALBUMOIDS. 


Albumins  proper. 

Serum  albumin,  egg  albumin,  lactalbumin. 
Globulins. 

Scrum    globulin,    egg    globulin,    lactoglobulin, 
cell  globulin,  vegetable  globulin. 
Coagulating  albumins. 

Fibrinogen,  myosin,  gluten  protein. 
Nucleoalbumins. 

Casein,  vitellins,  mucin-like  nucleoalbumins. 
Histones. 

Scomber-histone,  salmo-histone. 
Protamines. 

Salmin,  clupein,  stnrin,  scombrin. 

r  Coagulated  or  Modified  Albumin. 

Acid  and  Alkali  Albumins,  Albuminates. 
I,  Albumoses,  Peptones. 

Nucleoproteids. 

Nucleinic  acid   with  histone,  protamine,   etc. 
Hemoglobins. 

Hematin  with  histone. 
Glucoproteids. 

Combination    of    a    protein    and    carbohydrate 
group,  mucin,  mucoids,  phosphoglucoproteids. 

Collagen,  forming  gelatin,  glue. 
Keratin,  in  horn,  hair,  nails,  etc. 
Elastin,  elastic  tissue. 
Amyloid,  in  pathological  formations. 
Spongin,  in  sponge. 


GENERAL  REACTIONS  OF  THE  PROTEINS. 

The  various  substances  in  the  protein  group  respond  to  a 
number  of  reactions  which,  taken  together,  are  sufficient  to 
characterize  and  identify  the  bodies  in  question.  They  all 
contain  nitrogen  in  a  form  to  be  liberated  as  ammonia  when 


THE    PROTEIN    SUBSTANCES.  6/ 

the  dry  substance  is  heated  with  soda-Hme.  A  positive  result 
with  this  test  does  not,  of  course,  prove  the  presence  of  a  pro- 
tein compound,  since  all  ammonium  salts  and  amino  com- 
pounds in  general  respond  to  it;  but  with  a  negative  result 
proteins  as  well  as  these  other  compounds  are  certainly  ex- 
cluded. The  reaction  therefore  serves  as  a  preliminary  test 
in  the  examination  of  unknown  substances  for  proteins.  The 
test  may  be  easily  carried  out  and  is  delicate. 

Ex.  Mix  some  dried  albumin  or  some  wheat  flour  with  an  equal  bulk 
of  soda-lime  in  a  dry  test-tube.  Apply  heat  and  note  the  escape  of  am- 
moniacal  vapors  as  shown  by  the  odor  or  reaction  on  moist  litmus  paper. 
The  fixed  alkali  decomposes  the  protein  matter  very  quickly,  and  ammonia 
always  results. 

COAGULATION   TESTS. 

Many  of  the  protein  substances  undergo  a  peculiar  change 
known  as  coagulation  when  heated,  or  treated  with  certain 
reagents.  The  test  is  characteristic  of  most  of  the  bodies 
except  some  of  the  products  of  transformation.  This  coagu- 
lation is  usually  accompanied  by  precipitation,  that  is,  the  body 
is  thrown  out  of  solution  and  as  a  rule  can  not  be  restored  to 
its  original  condition.  But  there  are  cases  of  precipitation 
without  coagulation;  the  terms  must  not,  therefore,  be  used 
as  synonymous.  In  coagulation  proper  the  protein  body 
becomes  permanently  altered,  so  that  it  can  not  be  brought 
into  its  original  condition  again  by  addition  of  reagents  or  by 
other  means.  On  the  other  hand,  it  is  in  many  cases  possible 
to  throw  a  protein  body  out  of  solution  by  simply  adding  an 
excess  of  some  inorganic  salt,  without  at  the  same  time  pro- 
ducing any  decided  alteration  in  the  character  of  the  protein 
precipitate.  By  largely  diluting  with  water  the  precipitate 
may  be  brought  into  the  soluble  condition  again.  This  will 
be  illustrated  later  by  the  use  of  ammonium  sulphate  which 
behaves  in  a  characteristic  manner  with  different  proteins. 
Many  of  these  have  definite  precipitation  limits  with  the  sul- 
phate. That  is,  they  begin  to  separate  when  the  amount  of 
the  salt  reaches  a  certain  value,  and  precipitate  completely 
with  a  greater  concentration. 


68  PHYSIOLOGICAL    CHEMISTRY. 

Ex.  The  simplest  coagulation  may  be  shown  by  boiling  a  dilute  white 
of  egg  solution.  As  long  as  it  is  perfectly  neutral  coagulation  follows  at 
once,  but  in  presence  of  alkali  acid  must  be  added  to  the  point  of  neu- 
trality. This  behavior  finds  practical  application  in  the  ordinary  test  for 
serum  albumin  as  it  occurs  pathologically  in  urine.  The  precipitate  may 
be  redissolved  only  by  some  digestion  or  chemical  process  which  produces 
a  new  substance. 

Coagulation  by  Reagents.  By  the  addition  of  certain 
chemicals  many  of  the  protein  compounds  are  easily  thrown 
into  the  coagulated  condition.  Some  of  the  most  character- 
istic reactions  in  this  direction  are  shown  by  simple  experi- 
ments with  mineral  acids,  salts  of  heavy  metals  and  the 
so-called  alkaloid  reagents. 

Ex.  Among  the  acids  which  bring  about  coagulation,  nitric  acid  is  the 
most  certain  in  its  action  and  is  commonly  used  in  practical  cases  where 
it  is  desired  to  recognize  a  small  amount  of  serum  or  egg  albumin  in  solu- 
tion. The  test  may  be  made  by  adding  about  a  cubic  centimeter  of  strong 
nitric  acid  to  four  or  five  cubic  centimeters  of  white  of  egg  solution  and 
warming.  Coagulation  follows  at  once.  With  a  very  dilute  albumin 
solution  the  substance  separates  in  flakes,  while  with  a  strong  solution  a 
stiff,  jelh'-like  mass  may  result.  The  test  is  a  common  one  in  urine  analy- 
sis, but  must  be  conducted  with  certain  precautions. 

Ex.  Solutions  of  most  protein  substances  are  precipitated  by  addition 
of  alcohol  in  excess.  This  may  be  shown  by  mixing  white  of  egg  solution 
with  strong  alcohol,  the  latter  being  added  gradually  until  the  maximum 
of  precipitate  is  obtained.  With  dilute  alcohol  precipitates  are  usually 
not  secured,  and  besides  the  alcohol  precipitation  is  usually  not  a  perma- 
nent coagulation  as  in  the  above  case  with  the  acid. 

Ex.  Precipitation  by  Salts.  Some  of  the  salts  of  heavy  metals  give 
characteristic  precipitates  with  protein  solutions.  The  behavior  may  be  illus- 
trated by  adding  to  dilute  egg  albumin  solution  small  amounts  of  solution 
of  mercuric  chloride,  lead  acetate,  copper  sulphate  or  ferric  chloride.  The 
reagents  must  not  be  added  in  excess,  as  in  some  cases  this  causes  a  re- 
solution of  the  precipitate.  Similar  reactions  may  be  obtained  with  solu- 
tions of  most  of  the  heavy  metals,  but  the  salts  mentioned  are  often  used 
in  practice.  The  behavior  of  mercuric  chloride  or  corrosive  sublimate  as 
an  active  disinfecting  agent  depends  on  its  property  of  coagulating  the 
protein  matter  of  the  pathogenic  bacteria,  to  destroy  which  it  is  used. 
The  precipitates  may  be  formed  in  neutral,  acid  or  alkaline  solution  as  a 
rule,  and  chemically  may  be  regarded  as  salts  of  the  metals  used  as  pre- 
cipitants. 

Ex.  Precipitation  by  Alkaloid  Reagents.  In  acid  solution  the  protein 
bodies  are  very  generally  precipitated  by  addition  of  solutions  of  picric 
acid,    tannic   acid,   potassium-mercuric   iodide,   phosphomolybdic   acid   and 


THE    PROTEIN    SUBSTANCES.  69 

other  reagents  employed  in  the  detection  of  alkaloids.     The  precipitates  are 
voluminous,  and  in  most  cases  complete  in  presence  of  sufficient  acid. 
White  of  egg,  much  diluted,  may  be  used  in  illustration. 

The  above  reactions  may  be  explained  on  the  assumption 
that  the  proteins  here  act  as  pseudo-acids  or  pseudo-bases. 
In  perfectly  pure  solution  they  are  neutral  and  indifferent  to 
some  indicators,  but  the  addition  of  a  mineral  acid  imparts 
to  them  the  character  of  pseudo-ammonium  bases  which  yield 
precipitates  as  the  alkaloids  do  under  like  circumstances.  On 
the  other  hand,  with  salt  solutions  they  become  pseudo-acids 
and  form  now  insoluble  precipitates  of  complex  salts.  But 
it  has  been  shown  that  while  some  of  the  proteins  may  be  neu- 
tral to  litmus  they  may  at  the  same  time  be  quite  distinctly 
acid  to  phenolphthalein,  and  require  a  decided  amount  of 
decinormal  alkali  solution  to  give  a  reaction  by  displacing  the 
acid  in  combination  with  them.  This  is  taken  to  indicate  that 
they  should  be  looked  upon  as  true  bases,  rather  than  as 
pseudo-bases.  The  amount  of  acid  which  may  unite  with 
certain  proteins  has  recently  been  found  with  considerable 
accuracy,  and  becomes  in  some  degree  a  measure  of  the 
basicity.  In  other  cases  it  may  be  shown  that  they  have  a  true 
rather  than  a  pseudo-acid  character  and  unite  with  alkali  to 
form  real  salts. 

Behavior  with  Millon's  Reagent.  In  this  we  have  one 
of  the  most  characteristic  reactions  of  the  protein  bodies. 
Millon's  reagent  is  made  by  dissolving  mercury  in  twice  its 
weight  of  strong  nitric  acid,  completing  the  reaction  by  heat. 
The  solution  obtained  is  diluted  with  twice  its  volume  of 
water.  It  contains  a  little  nitrous  acid.  When  warmed  with 
white  of  egg  and  other  proteins  it  imparts  a  deep  red  color 
to  the  coagulum  produced  and  often  to  the  containing  solu- 
tion. The  reaction  is  common  to  benzene  derivatives  which 
have  a  hydroxyl  group  attached  to  the  nucleus,  and  is  given 
by  phenol  for  example.  The  reaction  in  the  protein  substances 
is  due  to  the  presence  of  the  tyrosine  group  in  the  complex 
molecule.     This  group  seems  to  be  present  in  all  protein  bodies 


70  PHYSIOLOGICAL    CHEMISTRY. 

with  the  exception  of  gelatin,  so  that  the  reaction  is  a  nearly 
universal  one.  The  protein  derivatives  which  still  contain 
the  tyrosine  complex  likewise  show  the  reaction.  Tyrosine  is 
represented  by  the  formula  Cr,H40H.CH2.CHNHo.COOH, 
and  w^ill  be  referred  to  later,  as  it  is  an  important  decomposi- 
tion product  of  proteins. 

Ex.  Test  the  behavior  of  Millon's  reagent  by  adding  some  to  white  of 
egg  solution,  milk  or  flour,  and  applying  heat.  The  characteristic  color 
appears  almost  immediately.  Its  depth  depends  somewhat  on  the  concen- 
tration of  the  protein  substance  used.  Presence  of  much  salt  interferes 
with  the  test  or  may  even  prevent  the  reaction. 

Ex.  Apply  the  same  test  to  weak  solutions  of  phenol,  salicylic  acid  and 
thymol.  Note  the  color  and  character  of  the  reaction.  These  bodies  all 
have  a  benzene  nucleus  with  hydroxy]  combination.  If  pure  tyrosine  is 
available  a  very  dilute  solution  may  be  employed  for  tests.  It  is  said  that 
I  part  in  looo  of  water  gives  a  distinct  reaction.  Hydroquinol,  resorcinol 
and  a-  and  i3-naphthol  give  likewise  decided  reactions,  but  the  colors  are 
orange  yellow. 

The  Biuret  Reaction.  This,  like  the  above,  is  a  protein 
test  depending  on  the  presence  of  certain  groups  in  the  com- 
plex molecule.  When  biuret  and  several  substances  of  related 
composition  are  mixed  with  an  excess  of  alkali  solution,  either 
sodium  or  potassium  hydroxide,  and  then  a  few  drops  of  a 
weak  copper  sulphate  solution  are  added,  a  blue-violet  to  red- 
dish-violet color  is  produced.  The  shade  depends  on  the  con- 
centration of  the  solution,  and  on  the  composition  of  the 
reacting  group.  It  has  been  shown  that  the  reaction  seems  to 
follow^  with  compounds  which  contain  two  groups  —  CONHg 
directly  united  or  joined  by  a  carbon  or  nitrogen  atom,  as  for 
example : 


CONH2 

CONH= 

/CONH. 
HN< 

^CONH= 

/CONH. 

H,C< 

\CONH= 

CO  •  CONHj 
1 
NH  •  CONH: 

Oxamide 

Biuret 

Malondiamide 

Oxaluramide 

The  combination  of  copper  and  alkali  with  these  bodies  has 
been  recently  studied  and  formulas  determined.  If,  in  place 
of  using  a  solution  of  one  of  these  compounds,  some  white  of 
egg  or  other  protein   solution   is   employed   the   same  color 


THE    PROTEIN    SUBSTANCES.  /I 

appears.  The  absorption  spectra  from  pure  biuret  and  egg 
albumin  solution  are  the  same,  which  shows  that  the  albumin 
must  split  off  this  group  under  the  influence  of  the  alkali  used. 
The  reaction  is  one  of  extreme  delicacy  and  may  be  employed 
for  the  recognition  of  traces  of  protein  compounds.  It  is 
used  especially  in  the  detection  of  peptone,  one  of  the  derived 
protein  substances.  Derivatives  of  simpler  nature,  that  is, 
the  products  of  the  decomposition  of  proteins,  do  not  give  the 
reaction.  It  is  therefore  of  value  in  following  the  course  of 
experiments  on  the  digestion  or  hydrolysis  of  proteins,  as  the 
reaction  disappears  with  the  breaking  down  of  the  last  protein 
complex. 

Nickel  salts  exhibit  an  analogous  behavior,  but  show  orange 
yellow  colors.  Cobalt  solutions  give  reddish  colors,  but  not 
very  strong. 

Ex.  Prepare  a  dilute  white  of  egg  solution  and  add  to  5  cc.  of  it  some 
solution  of  potassium  or  sodium  hydroxide.  Then  add  one  or  two  drops 
of  weak  copper  sulphate  solution,  or  enough  to  impart  the  characteristic 
color.  An  excess  of  the  copper  yields  a  precipitate  and  must  be  avoided. 
The  reaction  is  much  sharper  with  albumose  and  peptone  derivatives  than 
with  the  original  native  protein.  Repeat  the  test  with  solution  of  nickel 
sulphate.  The  test  is  obscured  by  the  presence  of  ammonium  salts,  which 
is  sometimes  a  matter  of  importance  in  practical  work. 

The  a-Naphthol  Test  of  Molisch.  In  the  chapter  on  the 
sugars  it  was  shown  that  a  very  marked  color  reaction  is 
given  by  mixing  a  few  drops  of  a  weak  alcoholic  solution  of 
a-naphthol  with  the  sugar  solution  and  then  adding  some 
strong  sulphuric  acid.  The  same  behavior  is  shown  by  solu- 
tions of  some  protein  substances,  which  indicates  that  they 
must  contain  a  carbohydrate  group  of  some  kind.  The  reac- 
tion depends  on  the  formation  of  furfuraldehyde  by  the  decom- 
position of  the  sugar  by  the  strong  acid.  This  furfuraldehyde 
combines  then  with  the  a-naphthol  to*  produce  a  deep  violet 
color,  the  reaction  being  similar  to  that  between  furfuralde- 
hyde and  aniline  acetate  described  in  the  pentose  test  in  a 
former  chapter. 


72  PHYSIOLOGICAL    CHEMISTRY. 

Ex.  To  a  few  cubic  centimeters  of  white  of  egg  solution  add  five  drops 
of  a  10  per  cent  solution  of  a-naphthol  in  alcohol.  Then  carefully  add 
three  or  four  cubic  centimeters  of  strong  sulphuric  acid,  which  sinks  below 
the  lighter  solution.  Note  the  color  at  the  zone  of  contact  and  through- 
out the  liquid  on  shaking.  Alkalies  change  the  color  to  yellow.  Thymol 
solution  is  sometimes  used  instead  of  a-naphthol.  This  gives  a  deep  red 
color.  These  furfuraldehyde  reactions  are  extremely  delicate,  and  appear 
in  a  great  variety  of  tests.  Their  general  character  and  importance  should 
therefore  be  recognized. 

The  Xanthoproteic  Reaction.  This  is  a  delicate  test, 
depending  on  the  formation  of  yellow  nitro  derivatives  of  the 
phenol  groups  in  the  protein  complex.  Similar  reactions  are 
given  by  many  simpler  organic  substances  where  nitric  acid 
is  mixed  with  them  and  heat  applied.  The  color  produced  by 
nitric  acid  in  contact  with  the  skin  is  due  to  the  same  general 
reaction. 

Ex.  In  illustration,  add  some  strong  nitric  acid  to  white  of  egg  solu- 
tion. On  application  of  heat  the  yellow  color  appears.  By  neutralizing 
with  ammonia  the  color  changes  to  orange  yellow. 

Make  a  similar  test  by  warming  some  phenol  solution  with  nitric  acid. 
In  this  case  a  nitro  phenol  is  formed.  Pure  phenol  and  strong  nitric  acid, 
it  will  be  recalled,  yield  trinitrophenol  or  picric  acid,  CoHj- OH  ■(N02)3. 
Add  ammonia  to  neutralize,  as  before. 

The  Lead  Hydroxide  Test.  The  protein  bodies  contain 
sulphur  which  may  be  removed  by  action  of  an  alkaline  lead 
solution,  or  alkaline  bismuth  solution*  or  mixture.  The  sec- 
ond reaction  has  some  importance  as  it  is  the  source  of  a  fallacy 
in  the  so-called  bismuth  test  for  sugar  in  urine.  In  presence 
of  albumin,  sulphide  of  bismuth  is  formed  in  place  of  the  re- 
duction product  indicative  of  sugar.  As  all  protein  bodies  con- 
tain sulphur  the  test  is  a  general  one.  It  may  be  made  as 
follows : 

Ex.  Produce  first  a  soluble  alkaline  compound  of  lead  by  adding  to  a 
few  cubic  centimeters  of  lead  acetate  solution  enough  strong  alkali,  sodium 
or  potassium  hydroxide,  to  form  a  precipitate  and  redissolve  it.  Then  add 
the  protein  substance,  white  of  egg  for  example,  and  boil.  A  brown  or 
black  color  appears  and  sometimes  even  a  precipitate  of  black  lead  sul- 
phide. Only  a  part  of  the  sulphur,  however,  may  be  separated  in  this 
simple  manner.  Another  portion  seems  to  be  much  more  firmly  combined 
in  the  protein  molecule. 


THE    PROTEIN    SUBSTANCES.  73 

The  reactions  which  have  just  been  explained  are  the  most 
important  and  characteristic  of  all  which  have  been  suggested 
for  the  recognition  and  identification  of  the  proteins.  Numer- 
ous other  reactions  are  known,  however,  which  are  easily- 
observed.  Several  of  these  are  color  tests,  depending  on  the 
formation  and  combination  of  furfuraldehyde,  but  they  need 
not  be  described  as  in  principle  they  do  not  differ  much  from 
the  Molisch  test. 

QUANTITATIVE   DETERMINATION   OF   PROTEINS. 

The  above  tests  serve  for  the  recognition  of  proteins  but  not 
for  their  determination,  and  for  the  latter  purpose  it  may  be 
said  further  that  no  one  method  is  perfectly  suited  to  all  cases. 
Many  of  the  simpler  protein  bodies  are  determined  by  com- 
plete coagulation,  followed  by  weighing  of  the  precipitate 
formed.  This  involves  several  operations,  all  of  which  must 
be  very  carefully  performed.  For  example,  a  pure  native 
albumin  in  solution  may  be  coagulated  by  adding  a  few  drops 
of  acetic  acid  and  boiling  thoroughly.  The  coagulum  is  col- 
lected on  a  weighed  paper  filter  or  in  a  Gooch  funnel,  thor- 
oughly washed,  dried  and  weighed.  Instead  of  drying  and 
weighing  the  precipitate  it  may  be  decomposed  according  to 
the  Kjeldahl  process,  in  which  the  nitrogen  is  converted  into 
ammonia  by  digestion  with  sulphuric  acid.  The  ammonia 
is  easily  separated  and  measured.  The  nitrogen  is  14/17  of 
it.  By  multiplying  the  nitrogen  found  by  the  factor  6.25  we 
obtain  the  original  protein  content  on  tHe  assumption  that 
these  bodies  contain  16  per  cent  of  nitrogen.  This  method  is 
now  commonly  followed  in  the  determination  of  crude  protein 
for  many  technical  and  scientific  purposes.  But  in  many  cases 
an  error  is  naturally  introduced  because  of  the  uncertainty  of 
the  factor;  6.25  is  the  mean  value  for  the  native  proteins  and 
the  closely  related  bodies. 


74  PHYSIOLOGICAL    CHEMISTRY. 

COMPONENT   GROUPS  IN   THE   PROTEIN   COMPLEX. 

In  the  study  of  the  protein  molecule  as  a  whole,  a  limit  is 
soon  reached  in  any  attempt  to  fix  its  composition,  but  much 
may  be  learned  by  observing  the  various  products  formed  in 
reactions  by  which  the  molecule  is  broken  down  under  the 
influence  of  different  agents.  Some  of  these  reactions  are  ap- 
parently largely  hydrolytic  in  character,  and  in  a  degree  may  be 
compared  to  the  decomposition  of  a  fat  by  superheated  steam. 
In  this  very  simple  case  glycerol  and  fatty  acids  are  obtained 
and  we  conclude  that  they  were  not  actually  formed  in  the 
process,  but  that  they  were  present  in  combination  in  the 
original  fat.  In  treating  protein  bodies  in  a  similar  manner 
or  in  subjecting  them  to  the  decomposing  influences  of  acids 
or  alkalies,  a  number  of  products  are  formed.  These  must 
be  either  results  of  peculiar  disintegration  and  subsequent 
synthesis,  or  they  must  represent  groups  in  some  way  existent 
in  the  original  complex.  The  latter  view  is  strengthened  by 
the  fact  long  observed  that  certain  products  result,  whatever 
the  method  of  decomposition.  Leucine,  for  example,  is  found 
abundantly  among  the  products  liberated  by  subjecting  protein 
to  the  action  of  superheated  steam,  hot  hydrochloric,  nitric  or 
dilute  sulphuric  acid,  concentrated  alkali  solutions,  bromine 
water  under  pressure,  or  to  prolonged  pancreatic  digestion. 
The  almost  necessary  conclusion  must  be  that  in  these  varied 
reactions  the  leucine  could  not  have  been  formed  from  smaller 
disintegration  groups,  but  must  have  been  set  free  from  some- 
thing holding  it  in  the  protein  complex. 

The  decomposition  reactions  are  therefore  considered  very 
important  as  suggesting  probably  the  component  groups  in 
the  large  molecule.  In  the  following  pages  a  few  of  the  most 
important  of  these  reactions  and  their  products  will  be 
described. 

Decomposition  by  Steam  under  Pressure.  By  pro- 
longed heating  of  protein  substances  with  water  certain 
changes   take  place,   even   below   a   temperature  of    ioo°    C. 


THE    PROTEIN    SUBSTANCES.  75 

FolloAving  the  coagulation,  which  appears  in  most  cases,  a 
gradual  hydration  and  solution  begins,  and  a  small  portion  of 
the  substance  is  brought  into  the  form  of  albumose  or  pos- 
sibly peptone.  At  a  higher  temperature,  that  is,  by  heating 
with  water  or  steam  under  pressure,  more  profound  changes 
take  place.  Ammonia  and  hydrogen  sulphide  are  split  off 
from  the  molecule  and  relatively  large  amounts  of  albumose 
and  peptone  are  formed.  If  the  temperature  is  high  enough 
the  reaction  extends  to  the  complete  destruction  of  the  mole- 
cule and  such  bodies  as  leucine  and  tyrosine  are  produced  in 
quantity. 

Effect  of  Alkalies.  Much  more  decided  changes  are 
noticed  when  the  protein  body  is  heated  with  alkali  solutions. 
Experiments  of  this  kind  were  long  ago  carried  out  by 
Schiitzenberger  and  have  since  been  frequently  repeated. 
Numerous  compounds  have  been  identified  among  the  decom- 
position products,  and  of  these  the  most  important  are  leucine 
in  quantity,  tyrosine,  ammonia,  carbonic  acid,  butyric  acid, 
formic  acid,  acetic  acid,  oxalic  acid,  aspartic  acid,  amino- 
valeric  and  amino-butyric  acid.  Barium,  potassium  and 
sodium  hydroxide  solutions  have  been  used  for  the  purpose. 
By  melting  the  dry  proteins  with  alkali  some  of  the  same 
products  are  formed,  especially  leucine  and  tyrosine. 

Effect  of  Acids.  Many  experiments  have  been  made  on 
the  decomposition  of  protein  bodies  'by  boiling  with  acids,  and 
particularly  with  strong  hydrochloric  acid,  the  hydrolyzing 
power  of  which  is  very  great.  The  most  important  of  the 
products  isolated  in  this  way  are  the  following: 

The  Hexone  Bases.  The  term  hexone  bases  has  been 
given  by  Kossel  to  a  group  of  bodies  which  occur  commonly 
in  the  decomposition  products  of  practically  all  the  protein 
substances.  We  have  here  arginine,  CeHj^N^Os,  lysine, 
C6H14N2O2,  and  histidine,  CcHgNgOs-  The  first  appears  to 
be  a  guanidine  derivative  of  amino-valeric  acid,  the  second  is 
diamino-caproic  acid,  while  the  third  appears  to  be  a  diamino 
acid  of  composition  as  yet  unknown.     The  isolation  of  these 


76  PHYSIOLOGICAL    CHEMISTRY. 

compounds  was  a  very  important  step  in  the  direction  of 
clearing  up  the  constitution  of  the  proteins,  inasmuch  as  some 
of  the  simplest  of  these  bodies,  the  protamines,  seem  to  con- 
sist almost  wholly  of  the  hexones.  ]\Iore  will  be  said  about 
this  relation  later.  The  hexones  are  soluble,  crystalline,  opti- 
cally active,  compounds. 

Leucine,  C6H13NO2,  a-amino-caproic  acid,  or  a-amino- 
isobutylacetic  acid.  This  has  been  already  mentioned  as 
found  abundantly  among  the  products  of  protein  decompo- 
sition by  other  agencies.  In  some  cases  it  appears  to  con- 
stitute 50  per  cent  of  the  reaction  products,  and  must  there- 
fore play  a  very  important  part  in  the  original  complex  mole- 
cule. Leucine  is  found  in  several  different  forms;  the  com- 
mon product  obtained  by  acid  hydrolysis  is  right  rotating  and 
shows  in  hydrochloric  acid  solution  [a]^^-l-  17.3°. 

Tyrosine,  CgHuXOo,  /'-oxyphenyl  amino-propionic  acid. 
This  appears  to  be  a  component  part  of  all  the  common  pro- 
teins, with  the  exception  of  gelatin,  and  from  some  of  them 
has  been  obtained  in  quantity.  As  already  mentioned  this  is 
probably  the  substance  which  reacts  commonly  with  ]\Iillon's 
reagent.  Tyrosine  is  but  slightly  soluble  in  water,  from  which 
it  crystallizes  in  bundles  of  fine  needles.  Its  solutions  are 
optically  active.  In  presence  of  hydrochloric  acid  [a]^  = 
—  8°  to  —  15°,  the  rotation  varying  with  the  amount  of  acid. 

Glycocoll  or  Glycine,  C2H5NO2.  amino-acetic  acid. 
Obtained  abundantly  from  gelatin  and  also  from  other  pro- 
teins. It  is  very  soluble  in  water  to  which  it  imparts  a  sweetish 
taste.     Insoluble  in  alcohol  or  ether. 

AsPARTic  Acid,  C4H-NO4,  amino-succinic  acid.  Slightly 
soluble  in  water,  the  solutions  being  right  rotating  at  the  ordi- 
nary temperature. 

Glutaminic  Acid,  C5H9NO4.  a-aminoglutaric  acid.  The 
acid  is  found  in  several  optical  modifications.  The  common 
form  is  but  slightly  soluble  in  water  and  is  right  rotating. 

Phenylamino  Propionic  Acid,  C9H11NO2,  phenylalanine. 
This   substance   resembles   tvrosine   closelv   in   structure   and 


THE    PROTEIN    SUBSTANCES.  77 

behavior  and  is  a  common  product  of  protein  decomposition. 
Slightly  soluble  in  water  and  has  a  sweetish  taste. 

Amino  Propionic  Acid,  or  Alanine,  C3H7NO2.  This  is 
in  a  sense  the  nucleus  substance  corresponding  to  tyrosine  and 
phenylalanine  just  referred  to.  It  is  a  soluble  product  rather 
widely  distributed  in  protein  bodies. 

Amino  Valeric  Acid,  CgHnNOg.  This  substance  is  ap- 
parently the  a  product.  It  is  usually  mixed  with  leucine  and 
is  separated  only  with  difficulty  from  this  body. 

a-PYRROLIDINE    CarBOXYLIC    AcID,    C5H9NO2,     (C4H7.NH 

COOH).  From  the  conditions  under  which  it  has  been  found 
this  interesting  body  is  supposed  to  be  a  primary  product.  It 
is  an  imino  not  an  amino  derivative.  It  was  first  obtained 
from  casein  and  later  from  other  proteins.  The  closely 
related  hydroxy-a-pyrrolidine  carhoxylic  acid  has  been  ob- 
tained from  gelatin. 

From  the  above  list  it  will  be  seen  that  the  amino  acids 
predominate;  most  of  them,  possibly  all,  are  the  a  compounds, 
and  as  will  be  shown  below,  they  make  up  a  very  large  portion 
of  the  whole  protein  molecule. 

Glucosamine,  C6Hii05(NH2).  This  appears  to  be  an 
important  constituent  in  some  groups  of  protein  bodies.  It 
has  been  obtained  in  quantity  from  the  glucoproteids  and  is 
possibly  present  in  small  amount  in  all.  It  is  usually  obtained 
as  a  salt,  hydrochloride  or  hydrobromide,  which  is  readily  sol- 
uble in  water  and  optically  active. 

Ammonia,  NH3.  This  is  always  found  in  relatively  large 
amount. 

Sulphur  Compounds.  Hydrogen  sulphide,  ethyl  sulphide, 
thiolactic  acid,  CgHeSOa,  cystin,  C6H12N2S2O4,  and  traces  of 
other  bodies  which  contain  sulphur  have  been  identified  in 
small  amount  among  the  decomposition  products. 

Carbonic  Acid  is  apparently  a  constant  derivative,  but  may 
appear  as  a  result  of  some  secondary  reaction.  Its  significance, 
therefore,  is  obscure. 

With  acids  other  than  hydrochloric  very  similar  reaction 


y8  PHYSIOLOGICAL    CHEMISTRY. 

products  are  secured.  It  will  be  shown  below  that  the  effects 
of  prolonged  tryptic  digestion  are  very  nearly  the  same  as 
observed  with  hydrochloric  acid.  This  is  a  point  of  the 
highest  practical  importance,  as  it  gives  us  some  insight  into 
the  complex  physiological  process.  And  in  peptic  digestion 
also,  W'here  very  weak  hydrochloric  acid  and  pepsin  are  em- 
ployed, essentially  the  same  products  result  provided  the  time 
of  the  action  be  made  sufficiently  long. 

RESULTANT  CHARACTER  OF  THE  PROTEIN  MOLECULE. 

While  the  various  decompositions  detailed  above  give  us 
some  insight  into  the  number  and  kind  of  groups  combined  in 
the  protein  complex,  they  do  not,  unfortunately,  show  us  much 
as  to  the  manner  in  which  these  groups  are  combined.  W'e 
are  not,  as  yet,  able  to  picture  to  ourselves  a  large  molecule  in 
which  the  leucine,  tyrosine,  aspartic  acid,  glycocoll,  and  so  on, 
are  united  to  form  a  molecule  with  the  general  properties  and 
molecular  weight  as  large  as  we  assign  to  even  the  simplest 
proteins,  but  a  step  has  been  made  in  that  direction  through  the 
synthesis  of  various  polypeptides  carried  out  by  Fischer  and 
Curtius,  who  have  succeeded  in  condensing  several  amino  acids 
into  one  molecule  w^ith  certain  properties  suggesting  those  of 
the  peptones.  These  bodies  w^ill  be  referred  to  in  a  later  chap- 
ter. Hofmeister  has  suggested  the  possibility  of  the  combi- 
nation of  amino  acids  in  large  groups  by  the  following  general 
scheme : 

—  NHCHCO  —  NHCHCO  —  NHCHCO  —  NHCHCO  — 


CH. 

1 

1 

CH, 

1 

(CHO3 

CHa-CH-CHa 

CeH.OH 

COOH 

CH=NH: 

Leucine 

Tyrosine 

Aspartic  Acid 

Lysine 

The  recognition  of  the  various  component  groups  suggests 
some  reasons  why  the  proteins  may  exhibit  acid  and  basic 
behavior  at  the  same  time.  Of  most  of  these  protein  com- 
pounds the  basic  character  is  the  more  pronounced  and  more 
readily  observed ;  that  is,  their  acid  combining  power.     Some 


THE    PROTEIN    SUBSTANCES.  79 

writers  consider  them  as  pseiido  bases,  and  hold  that  they 
become  actual  bases  only  through  peculiar  transformations 
effected  by  the  addition  of  acids.  But  careful  studies  of 
Osborne  have  shown  that  they  act  as  true  bases  and  unite 
directly  with  acids,  often  in  simple  proportions.  Many  of  the 
substances  which  we  commonly  call  pure  proteins  are  in 
reality  salts  of  the  true  bases.  These  salts  are  decomposed 
by  addition  of  weak  alkali  and  in  presence  of  litmus  a  neutral 
reaction  is  not  secured  until  after  the  addition  of  a  certain 
volume  of  the  alkali  solution.  Usually  a  still  further  volume 
of  alkali  must  be  added  tO'  produce  neutrality  with  phenol- 
phthalein. 

In  this  manner  several  of  the  proteins  seem  to  unite  with 
acid  in  the  proportion  of  one  molecule  of  protein  to  two  mole- 
cules of  a  monohydrogen  acid.  Because  of  the  very  great 
molecular  weight  of  the  protein  such  determinations  are  not 
easy,  however. 

The  presence  of  sulphur  hi  the  proteins  was  shown  by  a  test  referred 
to  some  pages  back.  Investigations  have  shown  that  sulphur  is  present 
in  at  least  two  kinds  of  combinations  in  the  protein  complex;  there  must 
be  at  least  two  sulphur  atoms  in  the  molecule.  Some  of  the  sulphur  is 
easily  separated  by  hot  alkali  solutions,  while  the  rest  of  it  is  not.  No 
part  of  this  element  appears  to  be  combined  in  oxidized  form,  that  is,  in 
the  condition  of  a  sulphite  or  sulphate.  The  sulphur  compounds  which 
have  been  obtained  in  protein  decomposition  are  such  as  may  be  derived 
from  a  breaking  down  of  the  cystin  group.  It  has  been  shown  that  cystin 
gives  up  its  sulphur  very  slowly  to  boiling  alkali,  and  then  only  in  part, 
as  sulphide. 

The  general  reactions  and  characteristics  of  the  protein 
bodies  having  been  discussed,  a  brief  description  of  the  more 
important  individual  substances  will  now  follow. 

TRUE    OR    NATIVE    ALBUMINS. 

In  the  scheme  of  classification  given  some  pages  back  the 
true  or  native  albumins  have  the  first  place.  The  best  known 
representatives  of  the  protein  group  are  included  here. 


So  PHYSIOLOGICAL    CHEMISTRY. 

ALBUMINS  PROPER. 

These  bodies  are  characterized  by  sohibihty  in  water  and  in 
weak  cold  acid  or  alkah  sohitions.  They  are  readily  coag- 
ulated by  heat  and  by  shaking  with  strong  alcohol.  Although 
usually  considered  as  amorphous,  the  albumins  have  been 
obtained  in  well  crystallized  form. 

The  characteristic  color  reactions  previously  referred  to  are 
all  given  by  the  true  albumins  and  they  are  precipitated  by 
ammonium  sulphate  or  zinc  sulphate  added  to  saturation. 
With  strong  sodium  chloride  precipitation  follows  only  after 
addition  of  acid. 

Serum  Albumin.  This  is  the  important  protein  body  of 
blood  serum,  of  which  it  constitutes  three  to  four  per  cent  by 
weight,  the  related  substance,  scrum  globulin,  making  up 
nearly  as  much.  While  closely  resembling  each  other,  it  is 
not  definitely  known  that  the  serum  albumins  of  different 
animals  are  identical.  In  blood  the  albumins  are  associated 
with  globulins,  fibrinogen,  mucoids,  salts  and  other  bodies,  the 
perfect  separation  of  which  is  practically  impossible.  The 
purification  of  serum  albumin  by  crystallization  is  not  easily 
carried  out  with  all  blood  serums ;  in  some  cases  the  formation 
of  crystals  is  slow  and  incomplete. 

Serum  albumin  contains  a  relatively  large  amount  of  sul- 
phur, about  two  per  cent  in  the  mean,  and  is  characterized 
further  by  a  high  specific  rotation.  The  values  which  have 
been  given  for  this  are  not  constant,  but  in  the  mean  are  about 

M^  =  -6o°. 

Crude  serum  albumin  is  now  an  article  of  commerce,  being 
made  in  large  quantities  from  blood  collected  at  the  slaughter- 
ing houses.  It  is  usually  mixed  with  globulin,  and  besides 
is  partly  insoluble  because  of  the  high  temperature  employed 
in  drying  it.  For  the  following  experiments  fresh  blood  must 
be  used. 

Ex.     Collect  blood  in  a  clean  vessel  and  stir  it  thoroughly  to  separate 
the  fibrin  and  part  of  the  corpuscles,  as  a  clot.     Some  of  the  corpuscles, 


THE    PROTEIN    SUBSTANCES.  Ol 

however,  remain  with  the  serum  and  may  be  separated  by  allowing  the 
latter  to  stand  in  a  tall,  narrow  jar,  or,  better,  by  rotating  the  serum  in 
a  centrifugal  machine.  Most  of  the  corpuscles  may  be  deposited  in  this 
way,  leaving  a  yellowish  liquid.  A  pure  white  serum  can  not  be  obtained 
because  a  little  of  the  hemoglobin  dissolves  from  the  corpuscles  and  re- 
mains in  solution.     With  this  prepared  serum  make  the  following  tests : 

Ex.  To  a  little  of  the  serum  add  finely  powdered  magnesium  sulphate  to 
saturation;  this  produces  a  precipitation  of  serum  globulin,  which  sepa- 
rates on  standing.  Pour  off  the  clear  liquid  and  add  to  it  powdered 
ammonium  sulphate,  which  gives  now  a  precipitate  of  albumin. 

Ex.  Mix  a  little  of  the  serum  with  two  or  three  volumes  of  water  in 
a  test-tube,  and  test  the  temperature  of  coagulation.  It  will  be  found 
near  70°  C. 

Lactalbumin.  Milk  contains  two  protein  substances,  the 
most  important  of  which  is  casein.  The  other  is  a  true  albu- 
min which  is  present  to  the  extent  of  about  one-half  per  cent 
in  cow's  milk.  It  resembles  serum  albumin  very  closely  but 
appears  to  have  a  much  lower  specific  rotation,  [a]^  ^  —  38"^. 

Egg  Albumin.  White  of  egg  contains  this  body  as  its 
characteristic  constituent  along  with  some  globulin  and  mucoid, 
and  traces  of  salts.  Common  albumin  reactions  are  usually 
made  with  white  of  egg  solution.  Although  this  substance  is 
always  described  as  a  true  albumin,  some  of  its  reactions  seem 
to  suggest  that  it  may  belong  to  the  group  of  glucoproteids, 
or,  at  any  rate,  may  contain  such  a  compound  in  relatively- 
large  amount.  On  heating  egg  albumin  with  weak  acid  glucos- 
amine is  split  off  and  in  quantity  sufficient  tO'  indicate  a  rather 
large  sugar  content  in  the  original  substance.  The  spe:ific 
rotation  is  much  lower  than  that  of  serum  albumin  and  may 
be  taken  at 

[a]  ^=-38°. 

which  is  about  the  value  for  milk  albumin.  Besides  this  dif- 
ference, egg  albumin  has  a  much  lower  coagulating  tempera- 
ture than  has  been  given  for  serum  albumin,  viz.,  56°.  Egg 
albumin  is  much  more  easily  coagulated  by  ether  than  is  serum 
albumin.  Egg  albumin  becomes  very  quickly  insoluble  when 
mixed  with  strong  alcohol.  From  serum  albumin  it  differs, 
further,  by  this  interesting  property.  When  its  solution  is 
7 


82 


PHYSIOLOGICAL    CHEMISTRY. 


injected  into  the  blood  circulation  it  passes  unchanged  through 
the  kidneys  into  the  urine;  the  same  thing  happens  when  large 
quantities  of  white  of  egg  are  eaten.  It  seems  to  escape  diges- 
tion in  this  latter  case  and  be  absorbed  in  pure  condition,  to  be 
later  discarded  by  the  kidneys.  These  various  points  of  beha- 
vior indicate,  then,  a  rather  marked  difference  between  the  two 
kinds  of  albumin. 

Ex.  So-called  pure  egg  albumin  may  be  obtained  in  this  way:  The 
white  of  egg  is  shaken  in  a  bottle  with  some  broken  glass  to  thoroughly 
break  up  the  membranes.  The  foamy  mass  is  filtered  through  fine,  unsized 
muslin,  and  to  the  filtrate  an  equal  volume  of  saturated  ammonium  sul- 
phate solution  is  added.  This 
produces  a  precipitate  of  globu- 
lin which  after  24  hours  is  fil- 
tered off.  Ammonium  sulphate 
in  this  strength  does  not  pre- 
cipitate the  true  albumin.  To 
this  filtrate  a  little  more  satu- 
rated ammonium  sulphate  is 
added  and  until  a  precipitate  or 
turbidity  just  begins  to  show. 
This  is  caused  to  disappear  by 
the  cautious  addition  of  water, 
a  few  drops  at  a  time.  Finally, 
acetic  acid  of  ten  per  cent 
strength  saturated  with  ammo- 
nium sulphate  is  added  until  a 
turbidity  again  appears,  and 
then  the  mixture  is  allowed  to 
stand  24  hours  in  a  cool  place. 
A  part  of  the  albumin  separates 
in  the  crystalline  form.  This  is 
collected,  redissolved  in  a  very 
little  cold  water  and  reprecipi- 
tated  with  ammonium  sulphate 
and  acetic  acid  as  before.  The 
crystals  are  collected  on  a  filter,  then  transferred  to  a  dialyzer  with  water 
lor  the  separation  of  the  sulphate  by  dialysis.  In  this  way  a  nearly  pure 
albumin  may  be  obtained  in  solution,  but  the  crystallized  substance  has 
not  been  secured  free  from  salts. 

White  of  egg  contains  in  the  mean  about  86  per  cent  of 
water,  13  per  cent  of  proteins,  0.6  per  cent  of  mineral  matters 
and  a  little  fat.     The  yellow  of  egg  is  a  substance  of  very 


Fig.  8.  Form  of  Graham  dialyzer  fre- 
quently used  in  purification  of  proteins. 
The  simple  parchment  tube  dialyzers  now 
obtainable  are  more  efficient. 


THE    PROTEIN    SUBSTANCES,  83 

different  composition.  The  water  present  amounts  to  about 
50  per  cent,  the  proteins  to  i6  per  cent,  the  fat  to  30  per  cent, 
or  more,  while  the  ash  is  about  i  per  cent.  The  fat  contains 
a  notable  quantity  of  lecithin. 

GLOBULINS. 

The  proteins  of  this  group  differ  from  the  albumins  mainly 
with  respect  to  solubility  in  water.  In  pure  water  they  are 
practically  insoluble,  but  they  dissolve  in  moderately  dilute  salt 
solutions.  On  diluting  a  globulin  solution  of  this  kind  precip- 
itation follows.  Globulin  solutions  Coagulate  by  heat  in  much 
the  same  manner  as  observed  with  the  albumins,  but  in  general 
they  become  permanently  insoluble  even  more  readily  than  do 
the  albumins. 

The  preparation  of  pure  globulin  is  even  more  difficult  than 
the  preparation  of  pure  albumin.  The  globulin  must  first  be 
separated  by  precipitation  with  some  salt;  as  the  salt  is  later 
removed  by  dialysis  the  globulin  remaining  becomes  insoluble, 
which  makes  further  treatment  difficult.  Globulins  are  not 
known  in  crystalline  condition. 

Serum  Globulin.  This  substance  makes  up  a  large  frac- 
tion of  the  protein  in  blood  serum,  amounting  tO'  nearly  as 
much  as  the  serum  albumin.  For  a  long  time  it  was  con- 
founded with  the  latter,  and  it  was  only  after  a  lengthy  series 
of  investigations  by  different  chemists  that  its  true  nature  was 
recognized.  This  globulin  may  be  discovered  easily  in  the 
serum  when  the  latter  body  is  diluted  with  water,  but  the  sepa- 
ration is  never  quite  complete  by  the  water  treatment  alone,  as 
a  portion  always  remains  in  solution.  By  salting  out  with 
ammonium  sulphate  to  half  saturation  or  with  magnesium  sul- 
phate completely  the  desired  end  is  reached. 

The  coagulation  temperature  of  serum  globulin  is  given  as 
75°  and  the  specific  rotation  as  [a]^  =  —  48°,  but  these  num- 
bers are  somewhat  uncertain,  especially  the  latter. 

Other  Globulins.  Several  other  bodies  are  described  as 
globulins.     The  most  important  of  these  is  the  so-called  cell 


84  PHYSIOLOGICAL    CHEMISTRY. 

globulin,  which  is  possibly  identical  with  serum  globulin.  This 
substance  has  been  obtained  from  different  organs,  from  the 
liver,  from  the  pancreas,  from  muscle  plasma,  etc.  Some 
of  the  globulins  described  as  cell  globulins  have  a  lower  coag- 
ulating temperature  than  the  true  serum  globulin. 

In  the  crystalline  lens  of  the  eye  a  body  has  been  long  known 
which  is  called  crystallin.  In  coagulation  temperature  and 
specific  rotation  this  crystallin  appears  distinct  from  serum 
globulin,  and  further,  it  seems  to  be  made  up  of  two  related 
substances,  a  and  ^  crystallins. 

Globulins  have  been  described  in  milk  and  egg  and  also 
in  the  vegetable  kingdom  under  the  name  of  pJiyfoglobulins  or 
phytovitelUns.  This  last  designation  indicates  that  they  may 
be  classed  under  the  head  of  the  nucleo-albumins,  with  which 
bodies  they  have  much  in  common.  In  this  connection  some- 
thing will  be  said  about  them  later. 

COAGULATING    PROTEINS. 

Several  extremely  important  substances  belong  in  this  group, 
which,  like  fibrinogen,  have  the  property  of  spontaneous  coagu- 
lation. In  nature  they  exist  normally  in  the  soluble  and  dis- 
solved form,  from  which,  under  certain  influences,  not  always 
well  understood,  they  pass  to  the  solidified  condition.  This 
coagulation  is  a  different  thing  from  that  produced  by  heat- 
ing to  a  high  temperature  or  by  the  addition  of  reagents; 
the  changes  in  the  latter  case  seem  to  be  more  profound.  We 
use  in  English  the  term  coagulation  to  describe  both  classes  of 
alterations,  which  are  really  of  a  very  diff'erent  character,  as 
will  appear  from  what  follows. 

FiBRixoGEX.  Blood  contains  a  peculiar  protein  body  in 
small  amount,  to  which  it  owes  its  property  of  spontaneous 
coagulation.  This  body  is  called  fibrinogen  and  the  product 
of  coagulation  is  known  as  fibrin.  The  nature  of  and  impor- 
tant factors  in  this  change  have  been  long  subjects  of  investi- 
gation and  discussion ;  it  can  not  be  said  that  the  matter  has 
been  fully  explained  in  all  its  bearings.     The  essential  points 


THE    PROTEIN    SUBSTANCES.  85 

of  what  is  known  will  be  given  in  the  chapter  on  the  blood. 

As  a  chemical  substance  fibrinogen  is  not  known  in  per- 
fectly pure  condition,  since  to  hold  it  in  soluble  form  various 
agents  must  be  added  to  the  blood.  But  the  -fibrin  formed, 
doubtless  through  ferment  action,  is  easily  obtained  and  its 
properties  are  well  established.  As  usually  prepared  it  is  a 
white,  elastic,  stringy  mass,  insoluble  in  water,  but  somewhat 
soluble  in  salt  solutions.  Like  other  proteins  it  undergoes 
true  coagulation  through  elevation  of  temperature  or  action 
of  various  reagents.  Fibrinogen,  as  prepared  by  salting  out 
from  plasma  at  a  low  temperature,  coagulates  when  warmed 
to  56°.  Its  specific  rotation  has  been  found  only  in  presence 
of  salt  or  alkali  and  varies  from  [a]i,  =  —  36°  to  — 53° 
according  to  the  nature  of  the  admixture  or  method  of  prepara- 
tion. It  undergoes  digestion  with  the  body  ferments  very 
readily  and  has  therefore  often  been  used  as  a  starting  point 
in  digestion  experiments. 

Myosin  and  Myogen.  The  living  muscle  plasma  contains 
a  number  of  protein  substances,  one  of  which,  at  least,  possesses 
the  property  of  spontaneous  coagulation  as  observed  in  the 
solidification  of  the  muscle  after  death.  At  one  time  the  term 
myosin  was  applied  to  this  body  and  it  was  supposed  to  be  very 
simple  in  nature.  Numerous  investigations,  however,  have 
shown  that  the  chemistry  of  the  muscle  proteins  is  compara- 
tively complex  and  that  the  results  of  experiments  do  not  well 
agree.  In  the  older  sense  this  myosin  was  assumed  to  be 
derived  from  a  preexisting  body,  myosinogen,  in  the  living 
muscle,  much  as  fibrin  is  considered  as  derived  from  fibrinogen. 
The  solidified  myosin  behaves  as  a  globulin,  which  may  be 
illustrated  by  the  following  experiment : 

Ex.  Free  muscle  (round  steak)  as  far  as  possible  from  traces  of  fat 
and  sinews,  and  then  thoroughly  disintegrate  it  by  passing  through  a 
sausage  mill.  Then  wash  it  repeatedly  with  cold  water  until  the  latter 
is  no  longer  reddened,  and  the  residue  appears  white.  This  is  placed 
in  a  ten  per  cent  solution  of  ammonium  chloride  and  allowed  to  remain 
about  a  day,  with  occasional  shaking.  Myosin  dissolves  in  the  ammonium 
chloride  and  is  found  in  the  filtrate  when  the  mixture  is   filtered.     Pour 


86  PHYSIOLOGICAL    CHEMISTRY. 

the  filtrate  into  twenty  times  its  volume  of  distilled  water,  which  causes 
a  precipitation  of  the  insoluble  mj'osin.  Allow  to  settle  and  wash  three 
times  by  decantation.  Collect  the  precipitate  and  observe  that  portions 
of  it  dissolve  readily  in  ten  per  cent  solutions  of  sodium  chloride  and 
ammonium  chloride,  or  in  a  o.i  per  cent  solution  of  hydrochloric  acid. 
The  solution  in  salt  is  precipitated  by  the  addition  of  more  to  saturation. 

By  this  treatment  with  the  dikite  ammonium  cliloride  solu- 
tion nearly  all  of  the  protein  of  the  muscle  plasma  may  be 
removed,  leaving  the  stroma.  It  is  now  pretty  generally  rec- 
ognized that  this  solution  contains  two  substances  instead  of 
one.  The  first  of  these  is  still  called  myosin,  and  is  said  to 
make  up  about  20  per  cent  of  the  plasma  protein,  while  the 
name  myogen  is  given  to  the  other,  constituting  80  per  cent 
of  the  soluble  protein.  Myosin  is  the  part  of  the  plasma  which 
coagulates  or  solidifies  the  most  readily  and  may  be  separated 
from  the  plasma  by  adding  ammonium  sulphate  to  make  28 
per  cent  of  the  solution.  On  filtering,  the  myogen  may  be 
separated  by  adding  ammonium  sulphate  nearly  to  complete 
saturation.  The  coagulation  temperature  of  myosin  is  given 
as  47°,  while  that  of  myogen  is  56°.  The  former  becomes 
quickly  insoluble  on  addition  of  alcohol,  while  myogen  seems 
to  be  partly  soluble  in  alcohol.  IMyosin-fibrin  and  myogen- 
fibrin  are  the  names  given  to  the  coagulated  forms  of  these 
bodies.  J\'Iore  will  be  said  of  these  relations  when  we  come 
to  consider  the  muscular  substance  as  a  whole. 

NUCLEO-ALBUMINS. 
This  group  contains  bodies  which  in  the  pure  state  are  rec- 
ognized as  acids.  They  are  called  nucleo-albumins  because  of 
the  earlier  fancied  resemblance  to  the  nucleo-proteids.  The 
characteristics  of  the  latter  group,  such  as  the  presence  of 
nucleinic  acid  and  the  xanthine  bases  among  the  decomposition 
products,  are  wholly  wanting  in  the  nucleo-albumins.  Both 
groups  contain  phosphorus,  and  in  both  cases  the  phosphorus 
is  separated  in  complex  combinations  on  digestion  with  pepsin 
and  hydrochloric  acid ;  the  character  of  the  phosphorous  com- 


THE    PROTEIN    SUBSTANCES.  8/ 

pound  separated  is  very  different  in  the  one  case,  however, 
from  what  it  is  in  the  other. 

The  free  acids  are  but  shghtly  soluble  in  water,  but  in  the 
salt  form  they  are  very  soluble  and  these  solutions  do  not  coag- 
ulate on  boiling,  as  shown  by  the  behavior  of  casein  in  milk. 
The  addition  of  weak  acids  to  these  salt  solutions  forms  pre- 
cipitates of  the  free  nucleo-albumin  acids.  From  very  weak 
solutions  the  precipitate  may  not  separate  until  after  heating. 
A  large  number  of  bodies  have  been  described  as  nucleo-albu- 
mins,  but  only  those  will  be  mentioned  here  w^hich  are  well 
known. 

Casein.  Of  all  the  nucleo-albumins  this  is  the  best  known 
and  most  important.  It  occurs  in  milk  as  a  neutral  calcium 
salt,  and  in  the  case  of  cow's  milk  makes  up  nearly  4  per  cent 
by  weight.  It  may  be  readily  separated  from  milk  by  the 
addition  of  a  'little  acetic  acid.  In  precipitating,  the  fat  is 
usually  carried  down  too,  but  may  be  removed  after  drying  by 
treatment  with  ether  or  petroleum  spirit.  Rennin,  the  peculiar 
enzyme  of  the  calf's  stomach,  causes  a  kind  of  coagulation  in 
casein  solutions;  if  lime  salts  are  present,  which  is  practically 
the  case  in  milk,  the  coagulation  extends  to  the  formation  of 
a  curd  or  cheesy  mass  which  is  very  characteristic.  The  first 
product  formed  by  the  rennin  is  known  as  paracasein  and  the 
curd,  or  cheese,  is  the  calcium  combination  of  this. 

Casein  was  formerly  considered  as  an  alkali  albuminate 
because  of  its  behavior  with  acids  and  alkali  solutions.  Many 
of  its  alkali  combinations  are  now  produced  in  a  technical  way 
as  by-products  in  the  butter  and  cream  industries.  Plasmon 
and  nutrose  are  apparently  sodium-casein  compounds.  These 
are  used  as  foods,  but  some  of  the  others  find  application  in 
other  directions.  Casein  forms  two  series  of  salts  with  cal- 
cium hydroxide  and  other  bases  and  the  amount  of  metal  in 
several  of  these  has  been  found  with  considerable  accuracy. 
Most  of  these  salts  form  opalescent  rather  than  perfectly  clear 
solutions.  The  addition  of  sodium  chloride  or  magnesium  sul- 
phate to  these  solutions  in  sufficient  amount  completely  precip- 


88  PHYSIOLOGICAL    CHEMISTRY. 

itates  the  casein.  Like  the  other  niicleo-albumins.  casein  leaves 
a  pseudo-nuclein  residue  on  digestion  with  pepsin  and  hydro- 
chloric acid. 

ViTELLiN.  \\'hile  white  of  egg  contains  essentially  albumin 
proper  and  globulin,  the  yellow  part  is  extremely  complex, 
containing  many  substances.  At  least  two  of  these  compounds 
hold  phosphorus  in  combination;  one  of  these  is  lecithin,  re- 
ferred to  earlier,  and  the  other  is  the  nucleo-albumin  called 
vitcUin.  The  separation  of  these  substances  from  each  other 
is  extremely  difficult.  Vitellin  is  not  soluble  in  water,  but 
dissolves  in  weak  alkali  solutions ;  on  digestion  wnth  pepsin  and 
hydrochloric  acid  it  yields  a  pseudo-nuclein  residue  which  con- 
tains iron  as  well  as  phosphorus.  The  name  hcuiatogen  has 
been  given  to  this,  and  it  is  considered  as  of  great  physiological 
importance  because  of  its  iron  content.  It  is  possibly  one  of 
the  parent  substances  of  hemoglobin. 

Vegetable  Nucleo-albumins.  Most  of  the  protein  bodies 
thus  far  referred  to  have  belonged  to  the  animal  kingdom,  but 
as  plant  constituents  fully  as  great  a  number  occur.  The  exact 
nature  of  some  of  these  is  obscure,  but  many  valuable  obser- 
vations have  been  made  by  Osborne  and  other  chemists  in  the 
last  few  years  which  have  cleared  up  some  of  the  points  in 
dispute.     Only  brief  mention  can  be  made  here. 

In  wheat  flour,  for  example,  four  or  five  protein  bodies 
appear  to  be  present.  The  most  abundant  of  these  is  called 
by  Osborne  glutcnin  and  makes  up  over  4  per  cent  of  the 
weight  of  the  grain.  Next  in  abundance  is  another  important 
compound  known  as  gliadin,  amounting  to  about  4  per  cent 
of  the  grain  weight.  These  two  proteins  unite  in  the  forma- 
tion of  gluten  which  is  essential  in  the  production  of  an  elastic 
dough,  which  on  leavening  yields  a  porous  and  light  bread. 
Gliadin  is  soluble  in  dilute  alcohol  and  forms  an  opalescent 
solution  wnth  water.  In  some  respects  it  resembles  a  globulin. 
In  its  behavior  with  weak  alkalies  glutenin  bears  some  resem- 
blance to  casein.  Wheat  flour  contains  also  a  true  globulin  in 
small  amount. 


THE    PROTEIN    SUBSTANCES.  89 

A  peculiar  protein  body  known  as  zein,  or  maize  fibrin,  is 
found  in  corn  meal.  It  is  soluble  in  alcohol  but  not  in  water, 
and  is  not  soluble  in  dilute  alkali  solutions.  Corn  contains 
also  three  globulin-like  bodies  and  one  or  more  substances  to 
be  classed  with  the  albumins  proper. 

Edestin  is  a  characteristic  crystalline  phytovitellin  found 
rather  widely  distributed,  but  most  readily  prepared  from 
hemp  seed  and  squash  seeds. 

Legumin  is  found  in  peas,  beans  and  related  seeds;  it 
belongs  to  the  group  of  nucleo-albumins,  but  in  its  solubility 
conditions  resembles  the  typical  globulins.  The  legumin  ob- 
tained from  vetches  does  not  coagulate  on  boiling.  On  boiling 
a  solution  of  pea  legumin  a  jelly-like  substance  is  formed. 

Other  Nucleo-albumins.  In  the  eggs  of  fishes  there  is 
found  a  peculiar  vitellin  called  ichthiilin,  which  has  been 
obtained  in  crystalline  form.  It  is  not  soluble  in  water,  but 
yields  a  clear  solution  with  weak  alkalies. 

In  cell  protoplasm  several  different  nucleo-albumins  are 
found.  These  bodies  contain  iron,  are  insoluble  in  water  in 
pure  condition,  but  with  alkalies  form  salts  which  are  readily 
soluble. 

TRANSFORMATION    PRODUCTS. 

The  protein  bodies  which  have  been  described  in  the  fore- 
going pages  are  natural  unmodified  substances  or  primary 
products.  We  have  now  to  consider  briefly  a. class  of  impor- 
tant protein  compounds  which  includes  secondary  or  modified 
substances  which  in  the  main  are  derived  from  the  native  albu- 
mins just  discussed.  These  modified  forms  may  be  obtained 
in  various  ways,  but  for  convenience  three  groups  of  transfor- 
mation products  may  be  made,  as  shown  below. 

COAGULATED    OR    MODIFIED    ALBUMINS. 

It  has  been  shown  already  that  white  of  tgg  dissolves  easily 
in  water.  The  solution  so  made  undergoes  a  change  when 
heated  or  when  treated  with  certain  reagents.     This  change  is 


90  niYSIOLOGICAL    CHEMISTRY. 

called  coagulation  and  the  resultant  product  is  so  essentially 
altered  that  it  may  no  longer  be  brought  into  the  original  form, 
or  a  similar  form,  by  any  known  means.  Some  of  the  condi- 
tions of  coagulation  have  been  explained  above  and  illustrated 
by  experiments.  While  the  simple  or  native  egg  albumin  is 
soluble  in  water  the  modified  product  is  insoluble.  It  is,  how- 
ever, soluble  in  weak  acids  or  alkalies,  but  is  insoluble  in  solu- 
tions of  neutral  salts.  It  follows,  therefore,  that  while  coagu- 
lation or  modification  of  a  native  albumin  always  follows  on 
heating,  precipitation  may  not  result.  This  depends  on  the 
reaction  of  the  mixture.  Coagulation  or  modification  on  the 
one  hand  and  precipitation  on  the  other  are  perfectly  distinct 
phenomena.  In  the  case  of  egg  albumin  in  solution,  for  ex- 
ample, a  precipitate  forms  on  heating  as  long  as  the  solution 
is  nearly  neutral.  In  presence  of  salts  the  precipitation  is  more 
complete.  But  if  the  original  solution  is  alkaline  modification 
of  the  albumin  takes  place  but  without  precipitation,  as  soluble 
alkali  albuminate  is  now  formed.  In  presence  of  acid  in 
proper  amount  soluble  acid  albumin  is  formed.  Although 
often  used  synonymously  the  terms  coagulation  and  precipita- 
tion have  here  distinct  meanings. 

The  exact  nature  of  the  change  which  takes  place  when 
native  albumins  are  heated  is  not  known.  Hence  the  terms 
used  in  describing  the  phenomenon  are  somewhat  indefinite. 
They  are  "  modified,"  or,  to  freely  render  a  German  expression, 
"  denatured."  To  bring  them  again  into  the  original  condi- 
tion is  not  possible.  White  of  egg  may  sometimes  be  mod- 
ified or  altered  without  becoming  opaque,  and  the  same  is  true 
of  clear  blood  serum.  In  both  cases  we  have  coagulation 
without  precipitation. 

ACID   AND    ALKALI    ALBUMINS. 

These  products  represent  the  most  important  forms  of  the 
coagulated  modified  albumin,  and  are  now  recognized  as  salts 
of  the  albumin  nucleus  acting  as  an  acid  or  basic  ion.  They 
are  usually  secured  by  the  action  of  acid  or  alkali  in  excess 
on  the  native  albumin,  usually  white  of  egg. 


THE    PROTEIN    SUBSTANCES.  9 1 

Alkali  Albuminates.  Strong  alkali  solutions  act  very  ener- 
getically on  white  of  egg  and  the  reaction  is  always  accom- 
panied by  some  decomposition  of  the  latter.  There  is  a  loss 
of  nitrogen  in  the  form  of  ammonia,  and  of  sulphur  as  hydro- 
gen sulphide.  The  reaction  with  lead  solution,  production 
of  lead  sulphide,  disappears  after  the  alkali  treatment.  The 
most  characteristic  product  of  alkali  action  on  native  albumin 
is  a  thick  jelly-like  mass  and  is  known  as  "  Lieberkuehn's  jelly." 
It  may  be  obtained  as  follows  : 

Ex.  Add  strong  sodium  hydroxide  solution  to  white  of  egg,  with 
constant  stirring,  until  a  thick  jelly  is  formed.  Too  much  alkali  must 
not  be  added  here,  but  just  enough  to  make  the  maximum  of  jelly.  This 
is  next  cut  into  small  pieces  and  washed  in  distilled  water  several  times 
until  the  lumps  are  white  throughout.  They  are  then  heated  with  fresh 
pure  water,  but  very  gently,  until  they  go  into  solution.  This  is  then 
filtered  and  the  filtrate  precipitated  by  acetic  acid,  avoiding  any  excess. 
The  precipitate  is  washed  with  pure   water. 

The  alkali  albuminates  are  nearly  insoluble  in  water  and 
dilute  salt  solutions.  The  treatment  in  the  above  experiment 
yields  finally  a  moderately  pure  albuminate  which  may  be  dis- 
solved by  addition  of  excess  of  weak  acid  or  alkali. 

Ex.  Use  some  of  the  albuminate  of  the  experiment  to  test  other  prop- 
erties. Dissolve  a  portion  in  weak  hydrochloric  or  sulphuric  acid  and 
observe  that  the  solution  does  not  coagulate  on  boiling.  An  acid  solution 
is  precipitated  by  addition  of  sodium  chloride  to  saturation,  and  it  is  also 
precipitated  by  adding  weak  alkali  to  the  point  of  neutrality.  When  this 
neutral  point  is  reached  more  alkali  brings  about  solution  again. 

The  formation  of  Lieberkuehn's  jelly  illustrates  the  produc- 
tion of  the  alkali  albumin  at  once  in  the  cold.  A  similar 
result  is  obtained  by  heating  some  white  of  egg  solution  for  a 
time  with  very  weak  alkali.    A  clear  solution  is  finally  obtained. 

Ex.  Dilute  white  of  egg  with  water  and  add  a  small  amount  of  N/io 
alkali  solution.  A  few  cubic  centimeters  will  suffice.  Keep  the  mixture 
at  a  temperature  of  about  40°  to  45°  on  the  water-bath  through  an  hour, 
and  then  test  some  of  it  by  boiling  in  a  test-tube.  It  should  not  coagulate. 
To  a  portion  of  the  clear  solution  add  a  few  drops  of  phenol-phthalein 
indicator  and  then  run  in  dilute  sulphuric  acid  to  neutralization.  A  pre- 
cipitate  forms   as   shown    above. 


92  PHYSIOLOGICAL    CHEMISTRY. 

Acid  Albumin.  According-  to  the  view  held  at  one  time  the 
solution  of  the  alkah  albuminate  in  water  yields  an  acid  albu- 
min on  acid  treatment.  But  the  weight  of  evidence  now  indi- 
cates that  the  group  in  the  albuminate  having  an  acid  function 
is  different  from  the  group  in  the  so-called  acid  albumin  which 
certainly  plays  the  part  of  a  basic  radical.  Although  the 
albuminate  and  the  acid  al1)umin  have  certain  points  in  com- 
mon, as  will  be  shown,  they  are  not  identical.  It  appears, 
however,  that  while  the  albuminate  may  not  be  converted  into 
acid  albumin  by  action  of  weak  acid,  the  opposite  conversion 
is  possible;  that  is,  weak  alkali  will  change  acid  albumin  into 
albuminate.     Some  simple  experiments  may  be  made  here: 

Ex.  Dilute  white  of  egg  with  four  voUimes  of  water,  take  25  cc.  of 
the  mixture,  add  5  cc.  of  0.2  per  cent  hydrochloric  acid  and  warm  it  on 
the  water-bath  for  about  two  hours  to  a  temperature  of  45°  C.  Then  care- 
fully neutralize  the  solution  with  dilute  sodium  hydroxide,  using  phenol- 
phthalein  as  indicator.  This  precipitates  insoluble  acid  albumin,  which 
can  be  washed  with  water  by  decantation.  It  is  essential  that  just  the 
right  amount  of  alkali  be  added  here;  an  excess  would  redissolve  the  pre- 
cipitated acid  albumin  with  formation  of  alkali  albuminate.  The  washed 
acid  albumin  can  be  used  for  a  number  of  tests. 

Ex.  Dissolve  a  little  of  the  washed  acid  albumin  in  water  by  the  aid 
of  weak  hydrochloric  acid,  and  note  that  the  solution  does  not  coagulate 
on  boiling.  Observe,  however,  that  the  addition  of  common  salt  to  the 
acid  solution  brings  about  precipitation.  The  same  thing  was  found  to 
be  true  with  the  solution  of  alkali  albuminate  in  acid. 

In  forming  acid  albumin  from  a  native  albumin  the  action 
of  the  weak  hydrochloric  acid  employed  is  much  less,  destruc- 
tive than  is  the  action  of  the  alkali  in  producing  albuminate. 
The  actual  modification  of  the  protein  molecule  is  much  less 
profound.  Nothing  is  split  off  as  is  the  ammonia  or  hydro- 
gen sulphide  in  the  other  case,  and  this  may  account  for  the 
observed  fact  that  the  acid  albumin  may  be  changed  into  albu- 
minate by  use  of  weak  alkali.  It  must  of  course  be  remem- 
bered that  a  stronger  acid  may  not  be  used  in  making  the  acid 
albumin,  since  here  too  the  reaction  may  become  destructive. 

Syntonin.  This  appears  to  be  an  acid  albumin,  resulting 
from  the  action  of  dilute  acids  on  muscle,  and  is  very  readily 


THE    PROTEIN    SUBSTANCES.  93 

formed  in  presence  of  the  ferment  pepsin.  The  name  is  often 
appHed  to  all  acid  albumins,  but  it  is  perhaps  preferable  to 
restrict  its  use  to  describe  the  product  from  muscle. 

Ex.  Free  the  muscle  part  of  meat  from  fat  as  far  as  possible  and  run 
it  through  a  sausage  mill  several  times  to  bring  it  to  a  fine  state  of  sub- 
division. Wash  this  chopped  mass  with  distilled  water  until  the  washings 
remain  clear.  Now,  to  about  5  gm.  of  the  moist  residue  in  a  small  flask, 
add  50  cc.  of  dilute  hydrochloric  acid,  containing  o.i  per  cent  of  the  true 
acid.  Warm  the  mixture  slightly  (to  35°  or  40°  C),  and  keep  at  this  tem- 
perature about  three  hours.  Then  filter  and  test  the  filtrate.  It  contains 
the  soluble  syntonin,  held  by  the  excess  of  weak  acid  used. 

Ex.  To  a  small  portion  of  the  filtrate  add  weak  caustic  soda,  which 
produces  a  precipitate  soluble  in  excess  of  the  alkali.  This  latter  solution 
contains  albuminate. 

Boil  another  portion  of  the  filtrate.  It  does  not  coagulate  directly,  but 
after  the  addition  of  common  salt  precipitation  follows. 

It  must  be  remembered  that  the  action  of  both  acids  and 
alkalies  on  the  native  albumins  may  easily  extend  beyond  the 
formation  of  the  simple  products  here  mentioned.  These  are 
merely  limiting  cases.  It  has  been  already  shown  that  by  more 
prolonged  action  various  products  of  disintegration  are  ob- 
tained and  the  substances  just  described  represent  the  first 
stages.  With  slightly  stronger  acids  or  alkalies  or  by  eleva- 
tion of  temperature  the  more  easily  separated  of  the  amino 
complexes  begin  to  split  off.  The  condition  of  stability  is 
only  relative.  With  molecules  as  large  as  these  it  may  even 
be  possible  to  separate  some  of  the  outlying  groups  without 
greatly  impairing  the  integrity  of  the  whole. 

ALBUMOSES  AND  PEPTONES. 

By  the  simple  treatment  with  weak  acids  or  alkalies  alone 
the  changes  in  the  native  protein  bodies  are  of  the  character 
described  in  the  last  paragraph.  But  in  presence  of  certain 
enzymes  further  modifications  are  reached  and  these  have 
received  the  names  of  albumoses  and  peptones  when  they  are 
produced  by  the  ferments  of  the  digestive  tract.  It  is  indeed 
true  that  these  substances  may  be  produced  in  fairly  large 
amount  by  the  simple  chemical  treatment  or  by  heating  the 


94  PHYSIOLOGICAL    CHEMISTRY. 

protein  substances  with  water  under  pressure.  But  the  names, 
in  practice,  are  usually  restricted  to  the  products  of  enzymic 
formation. 

Of  the  exact  nature  of  the  reactions  by  which  these  sub- 
stances are  reached  httle  is  known.  They  represent  the  very 
last  stages  in  the  process  of  breaking  down  complex  native  pro- 
tein bodies  which  still  give  the  characteristic  protein  tests. 
Further  disintegration  leads  to  bodies  which  are  no  longer  pro- 
teins, but  which,  like  the  amino  acids,  are  simply  constituent 
groups  of  the  complex  protein  molecule.  The  peptone  sub- 
stances represent  a  more  advanced  stage  of  modification  than 
do  the  albumoses.  In  both  groups  of  bodies  w^e  find  the  reac- 
tions with  the  alkaloid  reagents  and  with  the  precipitating 
metallic  solutions  in  most  cases  still  marked ;  the  biuret  reac- 
tion is  also  still  present.  But  for  the  peptones  we  find  lacking 
the  property  on  which  the  salting  out  processes  depend.  By 
adding  plenty  of  ammonium  sulphate  or  zinc  sulphate  it  is 
oossible  to  throw  the  albumoses  out  of  solution ;  the  peptones 
do  not  respond  to  this  treatment  and  in  other  points  also  they 
are  further  removed  from  the  original  proteins  than  are  the 
albumoses. 

But  it  must  not  be  understood  that  the  distinction  between 
the  two  groups  is  perfectly  simple  and  clear.  Unfortunately 
much  confusion  still  prevails  in  the  literature  of  the  subject 
and  an  elementary  presentation  which  is  satisfactory  and  con- 
sistent is  not  yet  possible.  In  this  chapter  only  a  brief  outline 
of  the  relations  now  generally  accepted  among  chemists  and 
physiologists  will  be  attempted,  while  in  a  following  chapter 
on  digestion  some  of  the  more  practical  details  will  receive 
consideration. 

Basis  of  Classification.  The  general  classification  of  these 
substances  commonly  recognized  is  that  of  Kiihne,  which  was 
elaborated  mainly  in  conjunction  with  Chittenden.  The 
scheme  has  been  enlarged  and  modified  somewhat  by  other 
workers  but  in  its  important  features  the  ideas  of  Kiihne  still 
hold  the  first  place.     In  the  weak  acid  as  well  as  in  the  enzymic 


THE    PROTEIN    SUBSTANCES.  95 

treatment  it  is  easily  seen  that  the  common  proteins  are  not 
homogeneous  or  symmetrical  bodies.  On  the  contrary  they 
seem  to  contain  two  great  groups  which  respond  very  differ- 
ently to  the  action  of  the  digestive  agent,  whether  acid  or  fer- 
ment. A  part  of  the  original  complex  appears  to  break  down 
rather  quickly  and  go  into  a  soluble  form;  while  a  second 
portion  resists  this  breaking  down  process  pretty  effectually  as 
far  as  weak  acid  and  pepsin  fermentation  is  concerned,  at  any 
rate,  and  in  subsequent  treatment  with  the  more  active  pan- 
creatic ferment  it  yields  products  different  from  those  derived 
from  the  first  group.  To  the  first  or  less  resistant  fraction, 
Kiihne  gave  the  name  hemi  group,  and  to  the  second  or  more 
resistant  portion,  the  name  anfi  group.  It  was  later  noticed 
that  most  protein  bodies  seem  to  contain  a  third  large  group 
which  in  the  subsequent  breaking  down  yields  a  sugar  of  some 
kind.  Hence  a  further  or  carbohydrate  group  may  be  as- 
sumed to  exist  in  the  native  protein  molecules,  or  in  most  of 
them,  at  least. 

Albumoses.  In  the  first  stage  of  the  action  of  the  acid 
and  ferment  on  the  protein  body  a  kind  of  acid  albumin  appears 
which  passes  by  continued  digestion  into  the  next  or  alhumose 
stage.  Different  albumoses  seem  to  be  derived  from  the  sev- 
eral native  proteins,  and  these  may  be  called,  in  general,  pro- 
teoses. Names  have  also  been  given  to  them  corresponding  to 
their  origin.  We  have,  accordingly,  fihrinoses,  caseoses, 
myosinoses,  glohulinoses,  and  so  on.  Several  degrees  of  albu- 
mose  digestion  are  recognized ;  that  is,  bodies  are  produced 
which  behave  differently  on  treatment  of  the  digesting  mixture 
with  precipitating  reagents,  and  we  have,  therefore,  primary 
and  secondary  albumoses.  The  secondary  albumose  stage  rep- 
resents a  more  advanced  condition  of  change  on  digestion  than 
does  the  primary  albumose.  Finally,  the  secondary  albumose, 
by  prolonged  contact  with  the  digestive  agents,  passes  into  the 
peptone  stage.  Some  idea  of  the  existence  of  these  three 
stages  of  change  may  be  obtained  from  the  following  experi- 
ment in  which  commercial  peptone  is  taken  for  illustration. 


96  PHYSIOLOGICAL    CHEMISTRY. 

This  is  a  substance  made  by  the  partial  digestion  of  fibrin,  gel- 
atin, serum  and  other  bodies  and  is  not  uniform  or  homoge- 
neous in  structure.  It  contains  representatives  of  the  several 
classes  of  derived  digestion  products. 

Ex.  Dissolve  about  5  gm.  of  commercial  peptone  in  50  cc.  of  water 
and  use  small  portions  of  the  solution  for  these  tests :  To  one  portion 
add  some  strong  nitric  acid ;  this  produces  a  precipitate.  To  a  second  por- 
tion add  a  little  copper  sulphate  solution,  which  gives  a  light  greenish 
precipitate.  To  a  third  portion  add  a  few  drops  of  acetic  acid  and  then 
some  potassium  ferrocyanide.  This  makes  a  turbidity  or  may  even  cause 
a  precipitate.  Now  to  the  remaining  and  large  portion  of  the  original 
solution  add  an  equal  volume  of  a  saturated  solution  of  ammonium  sul- 
phate. A  marked  precipitate  of  primary  albumosc  separates  and  may  be 
filtered  off  after  a  time.  When  the  liquid  has  all  passed  through  the  filter 
note  that  the  precipitate  may  be  easily  dissolved  by  adding  fresh  water, 
and  further  that  this  new  solution  is  not  coagulated  by  boiling.  Note  also 
that  the  solution  gives  a  good  biuret  reaction. 

Ex.  Use  the  filtrate  from  the  primarj'  albumose  precipitate  for  a  further 
test.  Add  to  it  powdered  ammonium  sulphate  to  complete  saturation,  that 
is,  as  long  as  the  powder  dissolves  on  thorough  shaking.  Then  add  five 
to  ten  drops  of  a  weakly  acid  solution  of  ammonium  sulphate  (which  may 
be  obtained  by  adding  to  10  cc.  of  saturated  ammonium  sulphate  solution 
five  drops  of  concentrated  sulphuric  acid).  This  last  treatment  with  the 
acid  ammonium  sulphate  gives  a  new  albumose  precipitate  which  after  a 
time  may  be  separated  by  filtration.  Save  the  filtrate  and  test  the  pre- 
cipitate as  follows :  Dissolve  it  in  fresh  water  and  test  portions  with  copper 
sulphate,  nitric  ?cid  and  the  potassium  ferrocyanide.  These  reagents  gave 
precipitates  with  the  original  peptone  solution,  but  yield  nothing  with  the 
solution  of  the  new  albumose,  which  is  called  secondary  albumose. 

Ex.  The  filtrate  from  the  secondary  albumose  may  finally  be  tested. 
Add  to  it  an  excess  of  concentrated  sodium  hydroxide  solution  and  then 
a  drop  of  dilute  copper  sulphate  solution.  This  gives  a  purple  red  biuret 
color,  showing  the  presence  of  a  soluble  product  not  precipitated  by  am- 
monium sulphate  in  excess.  This  soluble  product  is  the  peptone,  repre- 
senting the  last  stage  of  the  true  digestion.  This  peptone  gives  no  pre- 
cipitation reactions  with  the  reagent  used  above. 

The  first  of  these  fractions,  or  the  primary  albumoses,  may 
be  converted  by  further  acid  treatment  or  by  digestion  into 
secondary  albumoses  no  longer  precipitated  by  half  saturated 
ammonium  sulphate.  By  solution  in  water  and  addition  of 
alcohol  it  is  possible  to  separate  this  primary  albumose  into 
two  sub-fractions  which  are  pretty  well  characterized.     The 


THE    PROTEIN    SUBSTANCES.  97 

first  of  these  is  known  as  heteroalhumose  and  is  insoluble  in 
weak  alcohol,  while  the  second,  or  alcohol-soluble  portion,  is 
called  protalhumose.  The  heteroalhumose  belongs  to  the 
above  mentioned  anti  group  and  is  further  changed  only  with 
difficulty.  The  protalhumose  belongs  to  the  hemi  group.  It 
is  quite  soluble  in  water,  and  in  dilute  alcohol  even  more  sol- 
uble. By  prolonged  peptic  digestion  the  protalhumose  passes 
into  the  secondary  albumose  known  as  deiiteralhumose  A,  and 
then  into  peptone  B. 

These  two  primary  albumoses  contain  no  carbohydrate 
group,  since  they  give  no  reaction  with  the  Molisch  or  Adam- 
kiewicz  test.  The  heteroalhumose,  on  complete  splitting,  fur- 
nishes much  leucine  and  glycocoll,  but  little  or  no  tyrosine ;  the 
protalhumose,  on  the  contrary,  yields  a  large  amount  of  tyro- 
sine, but  little  leucine  and  no  glycocoll.  These  reactions  point 
to  essential  differences  in  structure. 

Like  the  primary  albumose  fraction,  the  secondary  fraction 
may  also  be  further  subdivided  and  chemists  have  recognized 
two  or  three  products  here  which  have  been  called  deuteral- 
bnmose  A,  deuteralhumose  B  and  deiiteralhumose  C.  But  the 
exact  relations  of  each  one  of  these  to  the  primary  products 
are  not  yet  clearly  established.  A  fourth  body  called  deuteral- 
humose Ba  has  also  been  described,  but  it  is  now  generally 
recognized  as  a  primary  product.  In  addition  to  these  four 
substances,  which  may  be  fairly  definite  chemical  individuals, 
authors  have  described  a  number  of  other  precipitation  frac- 
tions which  are  probably  mixtures.  Of  all  these  bodies  the 
deuteralhumose  A  and  the  deuteralhumose  B  are  the  only  ones 
for  which  the  conditions  of  precipitation  have  been  carefully 
worked  out. 

Peptones.  The  amount  of  real  peptone  formed  by  the 
pepsin  digestion  is  always  small ;  the  large  amount  of  peptone 
produced  in  the  body  is  a  consequence  of  the  action  of  the  pan- 
creas enzyme  known  as  trypsin.  The  peptone  of  gastric 
digestion  is  a  mixture  of  products  from  the  hemi  and  anti 
groups  and  has  been  called  amphopeptone.     The  term  anti- 


98  PHYSIOLOGICAL    CHEMISTRY. 

peptone  is  orenerally  applied  to  the  final  product  of  the  ener- 
getic pancreatic  digestion.  Amphopeptone  has  been  obtained 
as  a  yellow  powder,  very  soluble  in  water  and  very  hygroscopic. 
It  diffuses  pretty  well  through  parchment  and  has  a  sharp 
bitter  taste.  It  is  not  possible  to  salt  out  the  peptone  from 
solution,  but  the  alkaloid  reagents  give  precipitates,  which  are 
soluble  in  excess.  Precipitates  are  formed  by  solutions  of 
several  of  the  heavy  metallic  salts  also,  but  not  by  copper  salts. 

The  two  forms  of  amphopeptone  which  have  been  described 
are  known  as  amphopeptone  A  and  amphopeptone  B.  The 
first  is  insoluble  in  96  per  cent  alcohol  and  is  further  charac- 
terized by  giving  a  strong  reaction  with  the  Molisch  reagent 
which  relates  it  to  the  carbohydrate  group.  The  second  is  sol- 
uble in  96  per  cent  alcohol  and  does  not  give  the  Molisch  reac- 
tion.    Both  forms  give  a  strong  biuret  reaction. 

As  to  the  exact  nature  of  the  antipeptone  referred  to  above, 
there  is  still  much  uncertainty.  This  was  assumed  by  Kiihne 
to  represent  the  final  product  of  pancreatic  digestion,  and  it 
was  supposed  that  even  prolonged  digestion  would  not  change 
it  further.  It  was  found  later,  however,  that  various  amino 
acids  appear  here  in  considerable  quantity,  and  that  the  diges- 
tion may  be  carried  so  far  as  to  yield  a  product  which  no  longer 
gives  the  characteristic  biuret  reaction ;  that  is,  a  product  from 
which  ever5'^thing  of  a  really  protein  nature  has  disappeared. 
This  matter  will  be  more  fully  discussed  in  a  following  chapter. 
The  reactions  described  as  characteristic  of  antipeptone  are 
similar  to  those  for  the  amphopeptone  in  the  main.  A  good 
biuret  reaction  is  obtained  if  the  digestion  is  not  too  prolonged, 
and  the  alkaloid  reagents  give  precipitates  which  are  soluble 
in  excess.  Some  of  the  metallic  salts  precipitate,  but  copper 
sulphate  not. 

Formerly  many  attempts  were  made  to  show  the  relation  of 
these  digestive  products  in  tabular  or  diagram  form,  but  it  is 
now  known  that  most  of  these  diagrams  were  inexact  or  rep- 
resented more  than  is  actually  known.  One  of  the  latest  tab- 
ular arrangements  is  that  of  Hofmeister  which  shows  the  rela- 


THE    PROTEIN    SUBSTANCES. 


99 


tions  of  the  two  primary  albumoses,  three  secondary  products 
and  final  peptones,  and  their  behavior  toward  certain  reagents, 
( -j- ^  present,  —  =  absent): 


Precipitation 
Limits  with 
(NHJ.SG,. 
Per  cent  ot 
Saturation. 

0  " 

S-o 

C3   0 

>.8  0 

^1 

Pi 

C.2 
0  ^ 

0.3 
0  ri 

■S  rt 
c  '^ 

1^ 

"o 

0  u 

a 

0  0 

ll 

£2 
a  a. 

.2   3 
pi,  " 

Primary 

24-42 

Heteroalbumose. 

insol.  in  32 

+ 

weak 

+ 

weak 

_ 

+ 

products. 

Protalbumose. 

sol.  in  80 

+ 

strong 

+ 

strong 

— 

+ 

Deutero- 

54-62 

Thioalbumose. 

insol.  in  60—70 

+ 

+ 

+ 

+ 

_ 

Strong 

albumoses 

Albumose    poor    in 

sol.  in  60-70 

+ 

+ 

+ 

+ 

— 

weaker 

A. 

sulphur,  A. 

Deutero- 

70-95 

B  I  Albumose. 

insol.  in  35 

+ 

+ 

+ 



-p 

albumoses 

B  II  Albumose 

insol.  in  60-70 

+ 

+ 

+ 

weak 

strong 

+ 

B. 

(gluco). 

B  III  a-Albumose. 

sol.  in  80 

+ 

+ 

+ 

strong 

— 

— 

B  III  /?- Albumose. 

sol.  in  80 

+ 

+ 

+ 

strong 

— 



Peptomelanin. 

sol.  in  80-90 

? 

strong 

— 

— 

Deutero- 

100 

C  Albumose. 

sol.  in  60-70 

+ 



strong 



_ 

_ 

albumoses 
C. 

-|-  acid. 

Peptones. 

Cannot  be 

Peptone  A. 

insol.  in  96 

+ 

+ 

strong 

salted  out. 

Peptone  B. 

sol.  in  96 

+ 

+ 

+ 

— 

— 

In  the  formation  of  the  albumoses  and  peptones  from  native 
protein  molecules  a  large  amount  of  water  is  added;  roughly 
the  action  may  be  compared  to  the  hydration  of  starch,  pro- 
ducing malt  sugar  and  finally  glucose.  As  the  original  mole- 
cule is  very  large  the  percentage  amount  of  water  taken  up  in 
the  hydration  is  much  less  than  is  the  case  in  the  carbohydrate 
conversion.  It  is  also  very  interesting  to  note  that  with  the 
progress  of  the  hydration  the  amount  of  hydrochloric  acid 
which  may  be  held  by  the  product  increases ;  the  smaller  mole- 
cules in  the  aggregate  resulting  from  the  hydration  have  a 
much  greater  capacity  for  combining  with  free  hydrochloric 
acid  than  the  parent  substances  have.  This  question  assumes 
considerable  practical  importance  in  connection  with  the  sub- 
ject of  gastric  digestion  and  acidity  of  the  stomach,  as  will  be 
shown  later. 


lOO  PHYSIOLOGICAL    CHEMISTRY. 

It  must  be  remembered  that  the  various  commercial  products 
sold  as  "  peptones  "  may  contain  many  other  substances,  and 
may  be  quite  unfit  for  use  as  food  or  in  medicine.  \\'hile  in 
some  cases  a  considerable  portion  of  real  peptone  (with  albu- 
mose)  is  present,  in  others  the  main  constituents  are  decompo- 
sition products  formed  by  too  long  digestion  of  the  meat  or 
fibrin  with  the  acid  and  pepsin  mixture.  Some  of  these 
commercial  peptones  appear  to  be  formed  by  digesting  with 
weak  acid  under  pressure,  which  results  in  the  formation  of 
bodies  of  little  nutritive  value;  indeed,  it  is  likely  that  such 
products  are  distinctly  harmful  when  taken  into  the  stomach 
of  man. 

THE    PROTEIDS. 

The  term  protcid,  as  already  explained,  is  used  to  designate 
a  certain  group  of  protein  compounds.  This  use  is  a  per- 
fectly arbitrary  one  as  the  word  was  once  employed  to  describe 
all  the  bodies  discussed  in  this  chapter.  According  to  the  gen- 
erally accepted  modern  classifications  the  bodies  now  called 
proteids  are  compound  substances  in  which  a  true  or  native 
albumin  is  found  in  combination  with  some  other  group  which 
often  may  be  separated  as  such.  .In  the  table  given  earlier  in 
the  chapter  three  such  combinations  are  mentioned :  the  nucleo- 
proteids,  the  hemoglobins  and  the  gluco-proteids.  A  brief  de- 
scription of  each  group  will  here  follow. 

NUCLEO-PROTEIDS. 

These  proteins  are  important  as  making  up  a  large  part  of 
the  cell  nucleus.  In  treating  tissues  rich  in  cells  with  the 
pepsin-hydrochloric  acid  digestive  mixture  it  was  long  ago 
recognized  that  a  certain  portion  went  easily  into  solution, 
while  another  portion  was  always  left  undissolved.  This  res- 
idue was  called  nuclein  and  was  found  to  contain  all  the  phos- 
phorus of  the  original  protein.  If  in  place  of  the  pepsin 
mixture  some  other  hydrolyzing  agent  is  used  the  general 
result  is  similar:  a  separation  into  two  component  parts  takes 


THE    PROTEIN    SUBSTANCES.  lOl 

place,  and  one  of  these  parts  is  a  simple  native  protein  sub- 
stance and  the  other  the  nuclein  or  further  and  final  decompo- 
sition product,  niideinic  acid.  The  nucleo-proteids  are  there- 
fore described  as  combinations  of  native  albumins  with 
nucleinic  acid. 

In  breaking  down  the  complex  nucleo-proteid  it  appears  that 
several  stages  must  be  distinguished,  the  body  described  as  a 
"  nuclein  "  containing  still  some  native  albumin.  Finally, 
however,  the  residue  or  characteristic  part,  the  nucleinic  acid, 
is  left.  Although  many  investigations  have  been  made  there 
is  still  much  uncertainty  about  the  nature  of  this  acid.  In- 
deed, from  different  parent  substances  acids  of  somewhat  dif- 
ferent properties  have  been  obtained,  so  that  it  is  customary  to 
speak  of  the  nucleinic  acids.     These  will  be  considered  below. 

Like  the  native  proteins  already  described  the  nucleo-proteids 
are  coagulated  by  heat  and  by  acids.  They  are  soluble  in 
water,  salt  solutions  and  also  in  alkali  solutions.  By  means  of 
large  excess  of  salt  they  suffer  precipitation.  In  the  last  few 
years  nucleo-proteids  from  different  sources  have  been  studied, 
especially  from  yeast  cells,  the  thyroid  gland,  the  pancreas 
and  different  kinds  of  spermatozoa.  The  sperm  and  sperma- 
tozoa of  sea  urchins  and  fish  have  furnished  a  number  of  these 
substances  because  of  their  relative  richness  in  cell  structures. 
Thus  characteristic  products  have  been  obtained  from  the  sper- 
matozoa of  the  salmon,  the  mackerel,  the  sturgeon  and  so  on. 
These  appear  to  be  distinct  bodies,  but  more  exact  investiga- 
tions may  show  that  the  apparent  dift'erences  depend  on  foreign 
proteins  not  completely  separated  in  their  preparation. 

Protamines  and  Histones.  The  simple  native  albumins 
which  exist  in  combination  with  nucleinic  acids  in  the  nucleo- 
proteids  are  described  by  recent  investigators  as  protamines 
and  histones.  In  structure  these  bodies  are  probably  the  least 
complex  of  all  protein  substances.  They  do  not  exist  free  in 
nature  but  in  combination  with  nucleinic  acids,  hematin  or 
other  simple  "  prosthetic  group."  The  protamines  contain 
no  sulphur  but  are  very  rich  in  nitrogen  and  low  in  carbon 


102  PHYSIOLOGICAL    CHEMISTRY. 

as  compared  with  the  ordinary  proteins.  They  are  not  coag- 
ulated by  heat  and  do  not  give  the  Millon's  reagent  reaction 
or  that  of  Adamkiewicz.  The  biuret  reaction  is  marked 
and  the  alkaloid  reagents  produce  precipitates.  Some  of  the 
groups  in  the  common  proteins  are  therefore  wanting  in  the 
protamines.  Several  of  these  bodies  have  been  isolated,  par- 
ticularly from  the  nucleo-proteids  of  fish  spermatozoa  and  the 
names  given  to  them  suggest  their  origin.  Thus,  we  have 
saliiiiii,  sturin,  scomhrin  and  clupciii.  In  recent  analyses  the 
following  formulas  have  been  found  for  the  more  important 
protamines : 

Salmin    CsoHstNitOo 

Clupein  CsoHe^NiiOB 

Scombrin   Cs^HrsNieOs 

Stiirin    CwHriNiTOg 

^^'hen  warmed  with  weak  acid,  or  when  subjected  to  pan- 
creatic digestion,  they  yield  at  first  protoncs,  corresponding  to 
the  peptones  of  ordinary  digestion  and  finally  simpler  products, 
among  which  the  hexone  bases,  arginine,  lysine  and  histi- 
dine  predominate.  From  salmin,  for  example,  over  80  per 
cent  of  arginine  has  been  obtained.  In  some  cases  of  decom- 
position the  cleavage  into  the  hexone  bases  has  been  nearly 
quantitative,  which  is  an  important  step  toward  establishing 
the  empirical  formula  of  the  parent  protamine.  The  prota- 
mines appear  to  have  rather  marked  toxic  properties. 

The  histones  are  more  complex  bodies  than  the  protamines, 
and  possibly  contain  the  latter  as  a  component  part.  It  is  also 
possible  that  the  histones  represent  a  stage  in  the  development 
of  the  protamines,  since  while  the  former  are  found  in  imma- 
ture spermatozoa,  the  latter  are  commonly  obtained  from  the 
mature  organisms.  The  histones  are  bodies  which  bear  close 
relation  to  the  albumoses,  and  show  several  of  the  reactions 
considered  as  characteristic  of  the  latter.  They  are  water 
soluble  and  yield  a  precipitate  with  ammonia  which  is  insol- 
uble in  excess  in  presence  of  ammonium  salts.    A  pure  aqueous 


THE    PROTEIN    SUBSTANCES.  IO3 

solution  free  from  salt  is  not  coagulated  on  boiling,  but  with 
a  little  salt  present  coagulation  follows.  They  are  precipitated 
by  nitric  acid ;  the  precipitate  dissolves  on  heating  but  appears 
again  on  cooling.  They  yield  precipitates  with  the  alkaloid 
reagents  in  neutral  as  well  as  in  weak  acid  solutions.  Histone 
solutions  added  to  neutral  and  salt-free  solutions  of  several 
native  albumins  yield  complex  precipitates  which  contain  for 
one  molecule  of  the  histone  one  or  more  molecules  of  the  other 
protein.  This  is  an  important  reaction  as  it  suggests  the  build- 
ing up  of  a  complex  protein  molecule,  with  the  histone  as  a 
component  group. 

The  histones  are  strongly  basic  bodies  with  a  relatively 
large  content  of  nitrogen;  they  have  not  yet  been  isolated  in 
crystalline  condition.  They  give  the  biuret  reaction  and  the 
xanthoproteic  reaction,  but  contain  no  carbohydrate  group,  or 
phosphorus.  Several  of  them,  perhaps  all,  contain  iron,  which 
is  important. 

The  best  known  histones  are  these: 

Globin.  This  makes  up  about  96  per  cent  of  the  hemo- 
globin of  the  red  blood  corpuscle,  existing  in  combination  with 
the  iron-containing  constituent,  hematin. 

Scombron,  Salmon,  Arbacion.  These  are  peculiar  his- 
tones which  have  been  isolated  from  the  spermatozoa  of  the 
mackerel,  salmon  and  sea  urchin.  Preferable  names  are  scom- 
ber-histone,  salmo-histone,  etc.  The  on  termination  may  be 
reserved  for  the  pro  tones. 

Nucleo-histone.  This  name  was  given  to  a  product  sepa- 
rated from  the  thymus  glands  of  the  calf  and  was  one  of  the 
first  studied. 

The  Nucleinic  Acids.  The  occurrence  of  these  important 
compounds  in  combination  with  protamines  has  been  referred 
to  several  times  in  the  last  few  pages.  By  different  processes 
of  separation  a  number  of  these  acids  have  been  obtained  from 
various  cell  structures,  and  especially  from  yeast  and  fish 
sperm  or  spermatozoa.     The  results  of  analyses  lead  to  for- 


104  PHYSIOLOGICAL    CHEMISTRY. 

mulas  of  about  the  following  character  in  nearly  all  cases: 
C4oH..oX,40o-,P4.  These  acids  have  not  been  obtained  in 
crystalline  condition.  They  are  but  slightly  soluble  in  cold 
water,  but  soluble  in  weak  alkali  solutions  when  they  form 
salts.  A  number  of  salts  of  the  heavy  metals,  which  are  insol- 
uble in  water,  have  been  made  and  studied.  When  boiled  in 
aqueous  or  acid  solution  the  nucleinic  acids  break  up,  yielding 
finally  the  characteristic  basic  bodies,  long  known  as  the  xan- 
thine bases,  phosphoric  acid  and  certain  carbohydrates. 

The  nucleinic  acids  may  be  considered  as  esters  of  a  complex 
phosphoric  acid  derived  from  the  condensation  of  four  H5O5P 
(true  orthophosphoric  acid)  molecules.  The  various  decom- 
position products  just  referred  to  give  some  idea  of  the  organic 
groups  replacing  the  hydrogen  in  the  phosphoric  acid.  These 
substituting  groups  need  not  always  be  the  same,  hence  the 
existence  of  different  nucleinic  acids.  From  the  wheat  embryo 
an  acid  known  as  tritico-nucleinic  acid  has  recently  been  iso- 
lated and  thoroughly  studied.  The  formula  determined  from 
a  number  of  preparations  was  found  to  be.  with  great  proba- 
bility, C4iHr,iNj603iP4.  A  silver  salt  of  the  composition 
AgoC4iH-r,Ni603jP4  was  obtained  from  this,  and  on  complete 
hydrolysis  it  was  found  to  yield  one  molecule  of  guanine 
(C5H5N5O),  one  molecule  of  adenine  (C5H5N5),  two  mole- 
cules of  uracil  (C4H4N2O0),  three  molecules  of  a  pentose 
(C5H10O5),  phosphoric  acid  equivalent  to  four  molecules  and 
other  decomposition  products.  From  nucleinic  acids  of  animal 
origin  the  xanthine  (purine)  bases  have  been  found  in  other 
proportions.  The  importance  of  these  relations  will  appear 
later  when  the  origin  and  nature  of  uric  acid  (trioxypurine) 
is  considered.  The  uric  acid  of  the  urine  probably  comes  from 
the  complete  breaking  down  of  nucleinic  acids  of  cell  struc- 
tures, since  both  purine  and  pyrimidine  derivatives  are  found 
here,  and  both  lead  to  uric  acid. 

Xucleinic  acids  from  yeast  and  other  sources  have  found 
some  application  in  medicine. 


THE    PROTEIN    SUBSTANCES.  10$ 

HEMOGLOBINS. 

The  discussion  of  the  important  subject  of  hemoglobins 
may  properly  be  left  to  be  taken  up  with  the  study  of  the  blood 
in  which  they  are  contained.  The  term  is  used  here  in  the 
plural  since  from  different  kinds  of  blood  bodies  of  somewhat 
different  properties  have  been  obtained.  Hemoglobin  in  gen- 
eral must  be  classed  among  the  compound  bodies  because  it  is 
distinctly  made  up  of  two  characteristic  parts,  a  histone, 
already  referred  to,  and  hematin. 

GLUCO-PROTEIDS. 

We  have  here  a  group  of  bodies  containing  a  number  of 
important  members  about  which  our  knowledge  in  most  cases 
is  not  very  extended  or  exact.  As  the  name  indicates  the  pro- 
teins here  concerned  contain  a  carbohydrate  constituent  which 
may  be  recognized  by  its  reducing  properties  when  the  sub- 
stance in  question  is  warmed  with  a  weak  acid  and  afterwards 
treated  with  Fehling's  solution  in  the  usual  way.  The  carbo- 
hydrate group  separated  appears  to  be,  in  most  cases  at  any 
rate,  glucose  amine.  Familiar  illustrations  of  these  gluco-pro- 
teids  are  found  in  the  mucins  and  related  bodies  called  mucoids. 
As  a  class  these  substances  are  characterized  by  relatively  low 
nitrogen  and  high  oxygen  content,  due  to  the  presence  of  the 
carbohydrate  group.  The  amount  of  carbon  present  is  also 
lower  than  in  the  common  proteins.  Of  the  exact  nature  of 
the  albumin  combined  with  the  carbohydrate  little  is  known, 
because  in  separating  the  two  groups  by  acid  or  alkali  treat- 
ment the  protein  constituent  is  so  changed  that  no  safe  conclu- 
sion can  be  drawn  as  to  its  original  nature. 

The  gluco-proteids  behave  as  acid  bodies.  They  are  not 
coagulated  by  heat  alone,  but  heating  with  acids  or  alkalies 
produces  a  complete  alteration.  With  weak  acetic  acid  a  pre- 
cipitate is  in  most  cases  formed  which  is  not  easily  soluble  in 
excess. 

Mucins.     These  bodies   are   found   in   various    secretions. 


I06  PHYSIOLOGICAL    CHEMISTRY. 

especially  in  the  saliva,  bile,  vaginal  fluid,  tears,  nasal  mucus, 
etc.  The  amount  present,  however,  is  always  small  and  the 
separation  very  difficult  in  pure  condition.  The  mucin  of  the 
submaxillary  gland  is  probably  the  best  known. 

These  bodies  contain  one  of  the  complex  protein  groups, 
since  they  give  the  reaction  with  IMillon's  reagent,  the  xantho- 
proteic and  the  biuret  reactions.  They  are  only  slightly  solu- 
ble in  water  and  in  presence  of  alkali  produce  a  viscous  stringy 
liquid  which  is  extremely  characteristic,  even  in  great  dilu- 
tion. On  warming  with  dilute  alkali  the  viscous  condition 
disappears  through  formation  of  alkali  albuminate.  On  treat- 
ment with  strong  alkali  or  superheated  steam  a  peculiar  body 
is  formed  which,  from  its  discoverer,  is  known  as  Landwehr's 
animal  gum.  This  is  now  known  to  contain  the  protein  and 
carbohydrate  complexes;  after  diluting  with  weak  acid  and 
boiling  the  sugar  reaction  may  be  easily  obtained. 

The  mucins  are  much  more  resistant  than  the  nucleo- 
proteids  against  the  action  of  reagents  or  ferments,  but  they 
undergo  both  peptic  and  pancreatic  digestion  slowly.  In 
urine  the  identification  of  mucin  is  often  a  matter  of  impor- 
tance, as  it  is  frequently  mistaken  for  albumin.  The  detec- 
tion of  mucin  depends  on  the  behavior  with  cold  dilute  acetic 
acid  and  also  on  the  solubility  in  hot  water  after  precipitation 
with  strong  alcohol.  Albumin  is  permanently  coagulated  but 
mucin  not. 

Mucoid  Bodies.  These  substances  are  found  in  the  ten- 
dons, cartilage,  the  vitreous  body  of  the  eye,  the  cornea  and 
elsewhere,  and  are  closely  related  to  the  mucins.  They  have 
the  viscous  properties  of  the  latter  but  in  general,  in  concen- 
trated condition,  form  stiffer  jelly-like  masses.  The  cornea 
and  sclerotic  coat  of  the  eye  are  made  up  largely  of  mucoids 
and  collagen  dissolved  in  water. 

The  mucoids  from  tendon  have  been  the  most  thoroughly 
studied.  An  extract  is  made  by  prolonged  treatment  with 
weak  lime-water.  The  solution  is  precipitated  by  acetic  acid 
and  the  precipitate  taken  up  with  ammonia.     These  operations 


THE    PROTEIN    SUBSTANCES.  lO/ 

repeated  several  times  give  a  nearly  constant  product.  The 
analyses  shovv^  48-49  per  cent  of  carbon,  30  of  oxygen  and 
below  12  of  nitrogen.     A  small  amount  of  sulphur  is  present. 

In  cartilage,  along  with  collagen  and  albuminoid  bodies,  a 
very  important  mucoid  known  as  chondro-mucoid  is  found. 
This  has  a  composition  not  very  different  from  the  tendon 
product  just  given,  but  contains  over  2  per  cent  of  sulphur, 
part  of  which  is  in  peculiar  ethereal  combination.  This  ether- 
eal product  is  separated  by  cleavage  with  dilute  acids  or  alka- 
lies and  is  known  as  chondroitin  sulphuric  acid,  and,  according 
to  Schmiedeberg,  has  the  composition  C18H27NO14.SO3.  On 
hydrolysis  this  acid  yields  chondroitin,  C1SH27NO14,  and  sul- 
phuric acid;  the  chondroitin  furnishes  acetic  acid  and  chon- 
drosin,  C12H21NO11 ;  finally  further  hydrolysis  breaks  the 
chondrosin  down  into  glucoseamine  and  glucoronic  acid,  ac- 
cording to  the  same  author,  but  later  researches  seem  to  indi- 
cate that  the  cleavage  is  not  as  simple  as  suggested.  It  has 
been  shown  also  that  this  complex  acid  is  not  peculiar  to  carti- 
lage, but  is  found  in  many  substances  belonging  to  the  albu- 
minoid group  of  proteins  as  well.  Although  widely  distrib- 
uted the  physiological  importance  of  the  body  has  not  yet  been 
determined. 

In  addition,  mucoid  substances  have  been  recognized  in 
urine,  in  blood  serum,  in  white  of  e.gg  and  several  pathological 
transudates  in  small  amount. 

ALBUMOIDS    OR    ALBUMINOIDS. 

These  substances  differ  from  the  real  proteins  both  phys- 
ically and  chemically;  the  physical  differences  are,  however, 
the  most  pronounced  and  characteristic.  The  important 
bodies  grouped  here  contain  the  different  kinds  of  gelatin  or 
glue-forming  compounds,  the  horn  substances,  the  spongin  of 
the  sponge,  elastin  of  the  so-called  elastic  tissues  of  the  body 
and  other  substances  of  less  importance.  They  are  all  firmer 
and  harder  than  the  common  proteins  and  as  a  rule  quite  in- 
soluble in  water,  and  in  general  resistant  against  the  action  of 


I08  PHYSIOLOGICAL    CHEMISTRY. 

reagents.  While  by  prolonged  treatment  with  superheated 
steam  or  acids  or  alkalies  they  yield  most  of  the  cleavage  prod- 
ucts described  as  characteristic  of  the  albumins,  some  are, 
however,  lacking.  The  tyrosine  group,  for  example,  is  absent 
from  gelatin,  or  present  in  minute  amount  at  most. 

In  food  value  the  albuminoids  are  quite  distinct  from  the 
other  proteins.  ^lost  of  these  substances  are  so  insoluble  in 
the  digestive  fluids  that  really  no  importance  as  foods  could 
be  ascribed  to  them.  Collagen,  which  yields  gelatin,  has  a 
limited  food  value  of  a  peculiar  kind  which  will  be  referred 
to  below.  All  these  substances  serve  as  supporting,  connect- 
ing or  protective  tissues  in  the  body,  and  they  are  characterized 
necessarily  by  a  kind  of  permanence,  which  depends  on  insolu- 
bility in  the  first  degree.  With  increasing  age  of  the  body 
the  albuminoid  tissues  become  harder,  firmer  and  less  elastic. 

COLLAGEN. 

The  best  known  of  all  these  albuminoids  is  the  collagen,  or 
glue-forming  substance,  found  as  ossein  in  bone,  in  cartilage, 
in  the  fibrils  of  connective  tissue,  in  tendons,  in  fish  scales  and 
elsewhere.  This  substance,  wherever  found,  is  insoluble  in 
cold  water,  but  by  prolonged  heating  with  water  it  passes  into 
the  soluble  form  known  as  gelatin,  glutin  or,  in  impure  condi- 
tion, as  glue.  The  change  seems  to  depend  on  the  taking  up 
of  a  molecule,  or  more,  of  water.  At  the  present  time  it  is 
made  in  enormous-  quantities  from  slaughter  house  by-products 
and  according  to  its  purity  is  employed  for  different  purposes. 
When  made  by  hot  water  extraction  from  clean  bones  or  car- 
tilage it  is  used  as  an  adjunct  to  food  and  also  in  the  prepara- 
tion of  emulsions  for  photographic  plates  or  gelatin  paper. 
The  product  from  common  material  is  used  as  joiner's  glue. 

Gelatin  softens  and  dissolves  in  water  at  a  temperature 
above  30°.  But  this  solution  point  depends  largely  on  the 
treatment  to  which  it  has  been  previously  subjected.  By  long 
heating  with  water,  and  especially  under  the  action  of  super- 
heated steam  gelatin  gradually  breaks  down  into  the  usual 


THE    PROTEIN    SUBSTANCES.  IO9 

cleavage  products  of  the  proteins.  As  this  cleavage  pro- 
gresses a  point  is  finally  reached  where  the  mixture  no  longer 
solidifies  on  cooling;  a  permanent  liquid  solution  is  obtained. 
By  hydrolysis  with  acids  this  condition  is  much  sooner  reached. 
Many  bacteria  also  have  the  power  of  "  liquefying  "  gelatin, 
which  depends  of  course  on  their  ability  to  decompose  the 
complex  into  the  more  easily  soluble  amino  acids  and  other 
compounds. 

Among  the  final  cleavage  products  of  gelatin  easily  recog- 
nizable glycocoll  is  probably  the  most  abundant.  Leucine, 
alanine  and  various  other  amino  acids  are  found  in  smaller 
amount.  Like  other  proteins  gelatin  yields  in  peptic  or  tryptic 
digestion  bodies  which  have  been  called  gelatoses,  gelatin  pep- 
tones and  so  on.  These  resemble  but  are  not  identical  with 
the  true  peptones,  which  fact  has  some  bearing  on  the  long- 
discussed  question  of  the  food  value  of  gelatin.  Gelatin  is  not 
converted  into  true  protein  in  the  animal  body  and  for  this 
reason  cannot  wholly  replace  the  albumins  as  food.  But  to 
some  extent  it  has  the  power  of  protecting  the  so-called  circu- 
lating albumin  from  katabolism,  by  undergoing  destruction 
itself.  This  sparing  or  protecting  power  is  limited,  however, 
and  the  gelatin  substances  can  not  permanently  replace  the 
native  proteins  in  this  way. 

Experiments  to  Illustrate  Properties  of  Gelatin.  Dissolve  enough 
gelatin  in  hot  water  to  make  a  solution  of  about  one-half  per  cent  strength. 
Use  portions  of  this  for  tests : 

To  some  of  the  solution  add  a  solution  of  tannic  acid;  this  gives  a  buff 
colored  precipitate.  Gelatin  solution  is,  conversely,  employed  as  a  test  for 
tannic  acid. 

To  some  of  the  solution  add  an  excess  of  strong  alcohol ;  this  causes 
precipitation.     This  behavior  is  of  importance  in  the  estimation  of  gelatin. 

Use  some  of  the  solution  with  the  test  reagents.  Apply  Millon's  re- 
agent, the  biuret  test  and  the  xanthoproteic  test. 

Prepare  a  strong  solution  of  gelatin  in  hot  water.  To  some  of  this  add 
solution  of  potassium  dichromate  and  pour  the  mixture  out  to  cool  in  a 
thin  layer  exposed  to  sunlight.  This  treatment  produces  an  insoluble 
mass  which  is  not  attacked  by  hot  water.  This  property  finds  application 
in  photo-engraving  processes. 

To  more  of  the  strong  gelatin  solution  add  a  trace  of  alkali  to  neu- 
tralize any  acidity  and  then  some  formaldehyde.     On  evaporating  to  dry- 


I  lO  PHYSIOLOGICAL    CHEMISTRY. 

ness  a  hard  mass  is  obtained  which  is  quite  insoluble  in  water  hot  or  cold 
and  which  has  found  many  applications  in  the  arts. 

Gelatin  to  be  used  in  cooking  should  be  nearly  white  and 
should  dissolve  in  hot  water  to  form  a  practically  colorless, 
odorless  solution.  Inferior  gelatin  gives  off  a  bad  odor  when 
heated  wath  water. 

Isiiiglass  is  a  kind  of  collagen  made  from  the  swimming 
bladder  of  certain  large  fishes.  On  heating  with  water  it 
yields  a  peculiar  gelatin  which  dissolves  completely.  Com- 
mon isinglass  is  largely  used  in  clarifying  beer  and  wine,  while 
the  pure  white  varieties  are  employed  in  thickening  soups  and 
jellies. 

KERATIN. 

This  is  the  important  insoluble  substance  in  horn,  the  hoofs 
of  cattle,  finger  nails,  hair  and  feathers.  As  can  be  inferred 
from  the  conditions  under  which  it  exists,  it  is  not  easily  at- 
tacked by  water  hot  or  cold,  by  weak  acids  or  alkalies,  or  by 
digestive  fluids.  By  prolonged  action  of  hot  hydrochloric 
acid,  however,  it  undergoes  gradual  hydrolysis  and  cleavage 
wnth  formation  of  the  usual  amino  acids  and  other  products. 
Leucine  is  apparently  the  most  abundant  of  these  products,  as 
much  as  i8  per  cent  of  the  weight  of  the  horn  shavings  taken 
having  been  obtained  by  certain  investigators.  Other  import- 
ant cleavage  products  found  are  tyrosine,  a-aminoisovaleric 
acid,  aspartic  acid,  glutaminic  acid,  phenylalanine,  a-pyrroli- 
dine-carboxylic  acid,  glycocoll,  etc. 

All  keratin  bodies  contain  large  amounts  of  sulphur;  some 
of  this  is  easily  split  off  in  the  form  of  hydrogen  sulphide.  A 
large  part  of  the  sulphur  appears  to  be  present  in  the  complex 
body  cystin,  CCH12N2S2O4.  The  xanthoproteic  and  Millon's 
reagent  reactions  are  very  characteristic  with  horn  substance. 
The  behavior  of  a  drop  of  nitric  acid  on  the  finger  nail  is  well 
known.  The  reactions  with  alkaline  lead  solutions,  yielding 
lead  sulphide,  are  easily  obtained.  The  use  of  lead  salts  in 
hair  dyes  depends  on  this  behavior. 


THE    PROTEIN    SUBSTANCES.  I  I  I 


ELASTIN. 


Elastin  differs  from  keratin  mainly  in  its  higher  content  of 
carbon  and  low  sulphur  content.  In  their  behavior  toward 
reagents  they  are  much  alike.  Like  keratin,  elastin  can  be  dis- 
solved only  by  change  in  composition.  Leucine  is  produced 
in  large  amount  by  the  hydrochloric  acid  cleavage,  and  glyco- 
coll,  tyrosine  and  other  amino  products  in  smaller  amount. 
Subjected  to  peptic  and  pancreatic  digestion  elastin  is  slowly 
dissolved,  yielding  albumins  and  a  kind  of  peptone.  Most  of 
the  protein  reactions  may  be  obtained  from  elastin  after  bring- 
ing it  into  solution  with  alkali. 

AMYLOID  SUBSTANCE. 

This  is  a  body  Avhich  is  found  in  the  so-called  amyloid  de- 
generation of  the  liver  and  kidney.  It  is  particularly  charac- 
terized by  the  reddish  brown  color  it  assumes  when  heated 
with  a  solution  of  iodine  in  potassium  iodide.  The  analysis 
of  amyloid  shows  a  large  amount  of  carbon  and  some  sulphur. 
It  is  insoluble  in  cold  water,  but  partly  soluble  by  long  heat- 
ing. It  gives  the  usual  protein  reactions  when  brought  into 
alkaline  solution,  and  contains  also  a  complex  group  which 
yields  chondroitin  sulphuric  acid. 

FOOD  STUFFS. 

In  the  preceding  pages  the  individual  substances  used  as 
foods  or  occurring  as  essential  principles  of  the  animal  body 
have  been  briefly  discussed.  In  nature  these  compounds  do 
not  occur  in  the  pure  free  condition,  but  are  practically  always 
mixed  with  other  compounds.  Before  passing  to  the  subject 
of  digestion  it  will  be  necessary  to  have  some  idea  of  the  gen- 
eral composition  of  the  ordinary  foods  as  used  by  man.  This 
information  will  be  presented  in  tabular  form,  the  figures  being 
average  values  from  tables  of  Atwater.  The  fuel  values  are 
given  in  so-called  large  calories. 


I  12 


PHYSIOLOGICAL    CHEMISTRY. 


ANIMAL  FOODS. 


Water 

Protein 

Fat 

Ash 

Fuel  Value 

Percent. 

Per  Cent. 

Per  Cent. 

Per  Cent. 

in  Calories 
per  Pound. 

Loin  of  beef,  edible  portion... 

61.3 

19.0 

19. 1 

..0 

"55 

Flank  of  beef,  edible  portion.. 

59-3 

19.6 

21. 1 

0.9 

1255 

Ribs  of  beef,  edible  portion... 

57.0 

17.8 

24.6 

0.9 

1370 

Round  of  beef,  edible  portion. 

67.8 

20.9 

10.6 

I.I 

835 

Canned  corned  beef 

51.8 

26.3 

18.7 

4.0 

1280 

Canned   roast  beef 

5^-9 

25.9 

14.8 

1-3 

1 105 

Breast  of  veal,  edible  portion. 

68.5 

20.4 

10.5 

I.I 

820 

Leg  of  veal,  edible  portion. . . . 

71.7 

20.7 

6.7 

I.I 

670 

Leg  of  lamb,  edible  portion... 

58.6 

r8.6 

22.6 

1.0 

1300 

Leg  of  mutton,  edible  portion. 

55-0 

17.3 

27.1 

0.9 

1465 

Lean  ham,  edible  portion 

60.0 

25.0 

14.4 

1-3 

1075 

Fat  ham,  edible  portion 

38.7 

12.4 

50.0 

0.7 

2345 

Loin  of  pork,  edible  portion... 

50.7 

16.4 

32.0 

0.9 

1655 

Chicken,  edible  portion 

74.8 

21.5 

2-5 

I.I 

505 

Turkey,  edible  portion 

55-5 

21. 1 

22.9 

1.0 

1360 

Black  bass,  edible  portion 

76.7 

20.6 

1-7 

1.2 

455 

Catfish,  edible  portion 

64.1 

14.4 

20.6 

0.9 

"35 

Salmon,  edible  portion 

64.6 

22.0 

12.8 

1.4 

950 

Trout,  edible  portion 

77.8 

19.2 

2.1 

1.2 

445 

Oysters  

83.4 

8.8 

2.4 

1-5 

335 

Hens'  eggs,  edible  portion 

73-7 

13-4 

10.5 

1.0 

720 

Butter   

1 1.0 
31.6 

1.0 
28.8 

85.0 
35-9 

3-0 

3-4 

3605 

Cheese,  full,  American 

2055 

Lard,  unrefined 

4.8 

2.2 

94.0 

0.1 

4010 

Oleomargarin  

9.5 

1.2 

83.0 

6.3 

3525 

Gelatin  

13.6 

91.4 

0.1 

2.1 

1705 

In  the  above  table,  it  will  be  observed,  the  animal  foods 
contain  all  a  large  amount  of  water.  The  solids  consist  essen- 
tially of  proteins  and  fat.  In  the  vegetable  foods  the  water  is 
much  less;  in  most  of  them  carbohydrates  are  the  character- 
istic principles  present.  The  protein  is  generally  much  lower 
than  in  the  animal  foods. 


FLOUR  AND  MEAL. 

As  illustrating  the  composition  of  a  common  vegetable  food 
the  following  tests  may  be  made : 

Ex.  Boil  a  small  amount  of  wheat  flour  with  Millon's  reagent.  The 
red  color  produced  shows  presence  of  proteins. 

Ex.  Moisten  about  25  gm.  of  flour  with  water  and  work  it  into  a 
dough.  Then  hold  this  under  a  fine,  slow  stream  of  water  and  by  knead- 
ing between  the  fingers  slowly  work  out  a  portion  of  the  mass  as  a  thin 


THE  PROTEIN  SUBSTANCES. 
VEGETABLE  FOODS. 


113 


Corn,  whole -. 

Cornmeal  

Popcorn   

Oatmeal   

Rice    

Rye  flour 

Wheat  flour,  entire 

Wheat  flour,  California 

Wheat  flour,  general  average. 

White  bread,  wheat 

Whole  wheat  bread 

Crackers    

Beans,  dry 

Beans,  dry,  Lima 

Peas,  dry 

Peas,  green,  edible 

Corn,  green,  edible 

Cabbage,  edible  portion 

Egg  plant 

Potatoes,  edible  portion 

Squash,  edible  portion 

Apples,  edible  portion 

Bananas,  edible  portion 

Chestnuts,  edible  portion 

Hickory  nuts,  edible  portion. 
Peanuts,  edible  portion 


15.0 
12-5 

4.3 

7-3 
12.3 
12.9 
II. 4 
13-8 
12  o 
35-6 
38.4 

7-1 
12.6 
10.4 

9-5 
74.6 
75-4 
91-5 
92.9 

78.3 

88.3 

84.6 

75-3 

5-9 

3-7 

9.2 


«.2 

9.2 
10.7 
16. 1 

8.0 

6.8 
13-8 

7.9 
II. 4 

9-3 
9-7 
10.2 
22.5 
18.1 
24.6 
7.0 

3-1 
1.6 
1.2 
2.2 
1.4 
0.4 

1-3 

10.7 

15-4 
25.8 


u 


fe 


3-8 
1-9 
S-o 
7.2 

0.3 
0.9 
1.9 
1.4 
i.o 
1.2 
0.9 
8.8 
1.8 

I  5 
1.0 

0-5 
I.I 

0-3 
03 
0.1 

0-5 
o-S 
0.6 
7.0 

67.4 
38.6 


68.7 

75-4 
78.7 
67. 5 
79.0 

78.7 
71.9 
76.4 

75-1 
52.7 

49-7 
72.4 

59-6 

65-9 
62.0 
16.9 
19.7 
5.6 

S-i 
18.4 

9.0 
14.2 
22.0 
74.2 
II. 4 
24.4 


1.9 
1.0 
1.4 
0.9 
0.2 
0.4 
0.9 

0-3 
0.5 
1.2 
0.4 
4.4 

4-5 
1-7 
o-S 
I.I 
0.8 
0.4 
0.8 
1.2 
1.0 
2.7 

2.5 


<!S3 

0^ 


1.4 
1.0 

1-3 
1.9 
0.4 
0.7 
1.0 
0.5 
o-S 
1.2 

1-3 
1-5 
3-5 
4.1 
2.9 
1.0 
0.7 
1.0 

0-5 
1.0 
0.8 

0-3 
0.8 
2.2 
2.1 
2.0 


>  O  § 


1,610 
1,655 
1,875 
1,860 
1,630 
1,630 

1,675 
1,625 
1,650 
1,205 
1,140 

1,905 
1,605 
1,625 
1,655 
465 
470 

145 
130 

385 
215 
290 
460 
1,875 

3,345 
2,560 


milky  liquid.  This  is  largely  starch.  After  some  time  an  elastic  residue 
is  left  insoluble  in  water.  This  is  "  gluten "  and  is  the  chief  nitrogenous 
element  of  the  flour,  which  has  been  already  referred  to.  It  may  be  sepa- 
rated into  several  constituents. 

Ex.  To  about  5  gm.  of  flour  add  10  cc.  of  water,  shake  thoroughly  and 
allow  to  stand  until  a  nearly  clear  liquid  appears  above  a  white  sediment. 
Filter  the  liquid  and  test  for  sugar  by  the  Fehling  solution.  Boil  some 
of  the  residue  with  water  and  add  iodine  solution  as  a  test  for  starch. 

Ex.  To  about  5  gm.  of  fine  corn  meal  in  a  test-tube  add  10  cc.  of  ether. 
Close  the  tube  with  the  thumb  and  shake  thoroughly.  Then  cork  and 
allow  to  stand  half  an  hour.  Shake  again  and  pour  the  mixture  on  a 
small  filter,  collect  the  ethereal  filtrate  in  a  shallow  dish  and  evaporate  it 
by  immersion  in  warm  water.     A  small  amount  of  fat  will  remain. 

Action  of  Yeast  on  Flour.  The  following  experiment  is  intended  to  illus- 
trate the  work  done  by  yeast  in  leavening  dough: 

Ex.  Crumble  two  or  three  grams  of  compressed  yeast  into  15  cc.  of 
lukewarm  water  and  shake  or  stir  the  mixture  until  the  yeast  is  uni- 


114  PHYSIOLOGICAL    CHEMISTRY. 

formly  distributed.  Then  stir  in  enough  flour  to  make  a  thick  cream  and 
allow  to  stand  over  night  at  room  temperature.  In  this  time  fermenta- 
tion of  the  small  amount  of  sugar  in  the  flour  begins  and  the  "  sponge  " 
swells  up  by  the  escape  of  bubbles  of  gas.  At  this  stage  mix  in  uniformly 
and  thoroughly  enough  flour  to  make  a  stiff  dough,  using  for  the  purpose 
perhaps  25  gm.  Put  the  dough  in  an  evaporating  dish,  keep  it  for  an 
hour  or  more  at  a  temperature  of  30°  to  35°  C.  and  obser\-e  that  it  in- 
creases very  greatly  in  size,  from  the  continued  action  of  the  yeast  in 
liberating  bubbles  of  carbon  dioxide.  If  a  good  hot  air  oven  is  at  hand 
the  experiment  is  completed  by  baking  the  leavened  mass.  The  nature 
of  the  yeast  fermentation  will  be  explained  later. 

Milk.  In  milk  we  have  a  substance  in  which  all  the  essen- 
tial food  elements  are  present.  The  average  composition  of 
cow's  milk  is  given  in  this  table. 

Water    87.4 

Fat  3-5 

Sugar 4-5 

Proteins    3-9 

Salts  0.7 

lOO.O 

Human  milk  contains  more  sugar  and  less  protein  than  the 
milk  of  the  cow.     Details  of  this  will  be  given  later. 


SECTION    II. 
FERMENTS  AND  DIGESTIVE  PROCESSES. 

CHAPTER    VI. 

ENZYMES    AND    OTHER    FERMENTS.     DIGESTION. 

In  the  course  of  time  the  conception  of  fermentation  has 
undergone  many  changes.  The  notion  was  first  associated 
with  those  processes  in  which  a  bubbhng  or  boihng  condition 
without  apphcation  of  heat  was  observed,  and  later,  as  the 
most  famihar  kind  of  fermentation  was  more  closely  studied, 
this  phenomenon  was  found  to  be  due  to  the  escape  of  gas. 
This  escape  of  gas  came  finally  to  be  recognized  as  the  essen- 
tial feature  of  fermentation  and  many  operations  bearing  no 
relation  whatever  to  alcoholic  fermentation  were,  through  con- 
fusion of  ideas,  frequently  associated  with  it. 

The  real  fermentation,  which  follows  when  saccharine 
juices  are  exposed  to  the  air,  had  been  studied  in  a  way  from 
the  remotest  antiquity,  but  no  rational  attempts  at  an  explana- 
tion of  the  process  were  made  until  after  the  middle  of  the 
seventeenth  century,  when  the  relation  of  alcohol  and  the  gas 
to  the  destruction  of  the  sugar  seems  to  have  been  fully  recog- 
nized. Several  other  reactions  were  associated  with  the  alco- 
holic fermentation;  in  the  leavening  of  bread  the  production 
of  a  gas  was  recognized,  and  it  was  noticed  that  iii  the  changes 
going  on  in  the  animal  intestine  gases  were  also  liberated  fol- 
lowing the  digestion  of  foods.  Along  with  the  alcoholic  fer- 
mentation there  was  included  under  the  general  name  the  pecu- 
liar change  which  takes  place  when  the  wine  formed  from 
saccharine  liquids  was  allowed  to  stand  exposed  to  the  air. 
To  be  sure  no  gas  was  formed  in  this  action,  as  in  the  other, 
but  something  in  common  was  recognized.     In  both  cases  it 

115 


Il6  PHYSIOLOGICAL    CHEMISTRY. 

was  noticed  that  a  scum  formed  over  the  hquid  and  that  a 
small  amount  of  this  substance  was  capable  of  quickly  inciting 
similar  fermentation  in  more  saccharine  liquid  or  wine.  The 
nature  of  this  scum  became  in  time  the  subject  of  microscopic 
investigation  (by  Leuwenhock)  and  we  have  here  probably 
the  beginning  of  our  real  study  of  ferments.  It  was  in  1680 
that  Leuwenhock  recognized  that  this  scum  in  the  case  of 
beer  yeast  consisted  of  minute  globules  with  peculiar  proper- 
ties ;  the  full  value  of  this  discovery,  however,  was  not  gener- 
ally admitted  and  more  than  a  century  passed  before  any  great 
advance  was  made  by  others.  Lavoisier  toward  the  end  of 
the  eighteenth  century  gave  the  first  explanation  of  the  chem- 
istry of  alcoholic  fermentation,  as  he  was  able  to  point  out  the 
relation  of  carbon  dioxide  and  alcohol  to  the  parent  sugar. 
But  the  cause  of  the  action  was  not  much  discussed  and  just 
what  the  function  of  the  cells  or  globules  of  Leuwenhock  is 
remained  obscure  until  the  time  of  Pasteur. 

Before  taking  up  the  important  work  of  Pasteur  something 
must  be  said  of  discoveries  in  other  directions.  The  older 
conception  of  fermentation  was  widened  by  the  addition  of 
new  facts.  In  1780  Scheele  isolated  lactic  acid  from  sour 
milk  and  later  investigators  began  to  look  for  the  agent  re- 
sponsible for  the  production  of  this  acid.  About  1848  the 
probable  nature  of  an  organism  which  appeared  to  be  always 
associated  with  lactic  fermentation  was  pointed  out  by  Blon- 
deau.  Various  formulas  were  given  for  the  production  of 
lactic  acid  in  quantity,  but  it  often  happened  that  the  final 
product  was  an  entirely  different  substance,  viz :  butyric  acid. 
Butyric  fermentation  was  therefore  added  to  the  list  of  these 
peculiar  reactions,  and  various  speculations  were  advanced  to 
connect  the  different  phenomena.  Meanwhile  the  situation 
became  still  further  complicated  by  the  gradual  recognition  of 
a  new  group  of  reactions  which  exhibited  many  of  the  essen- 
tial features  of  the  alcoholic  and  acetic  fermentations,  and 
which,  therefore,  of  necessity  were  classed  as  ferment  reac- 
tions.    Several  chemists  had  observed  the  peculiar  behavior 


ENZYMES    AND    OTHER    FERMENTS DIGESTION.  11/ 

of  a  substance  produced  in  germinated  barley;  this  substance 
possessed  the  power  of  converting  starch  into  a  sugar  which, 
from  its  origin,  was  called  malt  sugar.  Payen  and  Persoz,  in 
1833,  succeeded  in  isolating  the  assumed  ferment  from  the 
sprouted  barley,  which  they  termed  diastase.  About  the  same 
time  it  was  recognized  that  saliva  contains  a  similar  starch- 
converting  agent  which  was  later  separated  and  called  salivary 
diastase  or  ptyalin.  The  seeds  of  the  bitter  almond  were  stud- 
ied by  several  scientific  men  and  Liebig  and  Wohler  isolated 
a  ferment  body  which  they  termed  emiilsin-.  This  has  the 
property  of  converting  the  glucoside  called  amygdalin  into 
glucose,  prussic  acid  and  oil  of  bitter  almonds,  or  benzoic  alde- 
hyde. As  the  saliva  was  found  to  contain  a  ferment  acting 
on  starches,  so  the  gastric  juice  was  recognized  as  active 
through  the  presence  of  an  analogous  body  called  pepsin  which 
acts  on  proteins. 

Most  of  these  discoveries  were  made  before  1840.  The 
ferments  in  the  bitter  almond,  in  sprouted  barley,  in  the  saliva 
and  in  the  gastric  juice  were  all  found  to  be  soluble  in  water. 
They  were  therefore  called  soluble  ferments  as  distinguished 
from  the  yeasts  and  the  ferments  of  acetic,  lactic  and  butyric 
acids,  and  from  the  conditions  of  their  action  Liebig  was  led 
to  formulate  the  first  general  theory  of  fermentation,  the  mo- 
lecular  vibration  theory. 

THEORIES   OF  FERMENTATION. 

Liebig's  Theory.  Liebig  advanced  and  maintained  for 
years  this  view :  A  ferment  is  a  chemical  substance  whose 
particles  or  molecules  exist  in  a  peculiar  state  of  vibration,  and 
in  contact  with  other  bodies  this  ferment  is  able  to  set  up 
similar  states  of  vibration  which  result  in  the  breaking  down 
of  the  bodies  mixed  with  the  ferment.  Ferments  were  con- 
sidered along  with  bodies  undergoing  putrefaction,  and  many 
such  substances  were  supposed  to  be  able  to  bring  about  real 
fermentations.  According  to  a  somewhat  older  view  ferments 
were  said  to  act  by  their  mere  presence;  that  is  they  exerted 


II  8  PHYSIOLOGICAL    CHEMISTRY. 

what  was  described  as  a  catalytic  action.  No  real  attempt, 
however,  was  made  to  define  more  closely  what  was  meant 
by  this  catalytic  or  contact  action. 

The  Theory  of  Pasteur.  The  real  nature  of  yeast  as  a 
vegetable  growth  had  finally  become  established.  With  this 
admitted  Pasteur  advanced  the  proposition  that  alcoholic  fer- 
mentation is  a  consequence  of  the  life  of  the  organism  in  con- 
tact with  sugar  and  away  from  the  air.  Alcohol  and  carbon 
dioxide  are  products  of  the  yeast  cell  metabolism  under  these 
conditions.  The  cell,  according  to  the  Pasteur  view,  must  be 
furnished  with  a  proper  supply  of  oxygen  and  this,  under  the 
fermenting  conditions,  it  takes  from  the  sugar,  giving  off  car- 
bon dioxide  as  an  oxidation  product  and  producing  alcohol  at 
the  same  time,  as  a  result  of  the  breaking  down  of  the  sugar 
molecule.  Fermentation  is  then  to  be  considered  from  a 
purely  biological  standpoint  with  alcohol  and  carbon  dioxide 
as  excretory  and  respiratory  products  respectively.  This 
Pasteur  theory  soon  found  favor  with  the  majority  of  scien- 
tific men  and  gradually  supplanted  the  mechanical  notion  of 
Liebig  w'hich  could  not  be  brought  into  accord  with  experience 
in  other  lines.  Although  the  Pasteur  view  that  the  yeast  pro- 
duces alcohol  only  in  absence  of  free  oxygen  w^as  shown  to  be 
incorrect  the  theory  commended  itself  as  otherwise  satisfac- 
tory and  tangible. 

Following  this  a  similar  explanation  was  offered  for  the 
action  taking  place  in  the  formation  of  acetic  acid,  lactic  acid 
and  butyric  acid.  Here  microorganisms  are  also  concerned. 
These  live  on  certain  substances  and  produce  others  as  meta- 
bolic excreta.  As  to  the  mechanism  of  this  metabolism  we 
know,  of  course,  nothing;  to  describe  the  products  formed  as 
excreta  is  perhaps  not  really  warranted  by  what  is  actually 
known.  It  must  be  remembered  then  that  the  term  is  used 
in  a  broad  and  general  sense  only  to  indicate  some  kind  of  a 
metabolic  product. 

The  w^ork  of  Pasteur  gave  an  enormous  impetus  to  the  study 
of  the  common  fermentations,  but  it  was  evident  that  this  bio- 


ENZYMES    AND    OTHER    FERMENTS DIGESTION.  II 9 

logical  explanation  was  of  no  value  in  accounting  for  the 
changes  produced  by  the  active  agents  described  as  diastase, 
pepsin,  emulsin,  etc.  These,  it  was  pointed  out,  are  as  truly 
"  ferments  "  as  are  yeast  and  the  mother  of  vinegar.  To 
avoid  confusion  it  became  customary  to  speak  of  the  organised 
and  unorganized  ferments,  or  the  insoluble  and  soluble  fer- 
ments. The  term  enzyme  was  later  applied  to  these  soluble 
unorganized  agents  of  change,  but  this  new  expression  did 
nothing  toward  explaining  the  difficulty  or  toward  relating 
the  two  classes  of  ferments. 

Certain  scientists  from  the  start,  however,  refused  to  admit 
any  fundamental  difference  between  the  work  of  the  yeast 
ferment  on  the  one  hand  and  that  of  bodies  like  diastase  on 
the  other.  Even  after  the  biological  theory  of  Pasteur  had 
become  current  Berthelot,  Hoppe-Seyler  and  other  chemists 
of  prominence  maintained  that  the  living  cell  ferments  are 
active  because  they  secrete  soluble  or  enzymic  bodies.  In  the 
one  case  the  actual  "  fermentation  "  takes  place  within  the 
cell,  as  appeared  to  be  the  fact  with  yeast;  in  other  cases  en- 
zymes are  produced  by  cells  and  thrown  off  to  do  their  work 
elsewhere.  This  is  true,  for  example,  in  the  stomach  where 
certain  groups  of  cells  produce  the  active  ferment  pepsin  which, 
however,  does  its  work  of  dissolving  coagulated  protein,  or 
digesting  it,  outside  the  cells  themselves.  In  germinating 
barley  the  living  cells  secrete  diastase  which  may  be  leached 
out  and  used  to  digest  starch  of  other  grains.  If  not  leached 
out  the  diastase  gradually  digests  the  starch  of  the  barley  ker- 
nel itself,  unless  the  action  be  checked  by  heat  or  other  means. 

The  Work  of  Buchner.  In  principle,  therefore,  the  two 
kinds  of  ferment  action  were  held  to  be  alike,  but  although 
many  attempts  were  m.ade  no  chemist  succeeded  in  isolating 
the  assumed  enzyme  from  the  active  cells.  Repeated  failure 
in  this  direction  only  served  to  strengthen  the  belief  of  the 
advocates  of  the  vital  theory  according  to  which  alcoholic  and 
similar  fermentations  by  fungi  are  processes  which  cannot  be 
thought  of  dissociated  from  the  function  of  life  itself.     But 


I-O  PHYSIOLOGICAL    CHEMISTRY. 

finally  the  problem  was  solved  by  the  German  chemist  Buch- 
ner,  who  in  1897  succeeded  in  isolating  the  active  enzyme  from 
yeast  cells  and  in  quantity  too.  This  enzyme  he  called  ^73;- 
mase;  it  was  found  to  be  as  active  as  the  yeast  itself  and  to  do 
all  that  could  be  expected  of  yeast.  It  has  since  been  pro- 
duced on  the  commercial  scale  in  the  endeavor  to  supplant  the 
use  of  yeast  in  practice.  More  recently  it  has  been  shown  that 
other  ferment  cells  secrete  enzymes  and  it  is  possible  that  all 
the  so-called  organized  ferments  work  in  this  way;  but  the 
isolation  of  the  soluble  active  principle  seems  to  be  very  diffi- 
cult in  most  cases. 

All  this,  however,  affords  us  no  real  insight  into  the  nature 
of  the  ferment  process.  We  have  as  yet  no  satisfactory'  theory 
as  to  how  these  active  chemical  principles  behave  in  the  break- 
ing down  of  other  organic  substances.  It  has  not  been  found 
possible  to  prepare  any  enzyme  in  a  condition  of  even  approxi- 
mate purity  and  all  analyses  made  of  such  substances  are 
doubtless  wide  of  the  truth.  These  analyses  appear  to  show 
that  the  enzymes  are  of  protein  character,  but  the  impurities 
in  the  products  analyzed  may  be  responsible  for  this  indication. 
With  this  lack  of  knowledge  regarding  the  chemical  composi- 
tion of  the  ferments  it  is  naturally  impossible  to  offer  a  chem- 
ical explanation  of  how  they  act.  It  is  the  effects  only  that 
we  are  familiar  with  and  all  our  classifications  are  practically 
based  on  what  the  ferments  can  do  rather  than  on  what  they 
are.  It  is  known  that  all  ferments  are  destroyed  by  heat  and 
by  the  action  of  even  rather  weak  acids  and  alkalies.  In  this 
they  resemble  the  cells  that  produce  them.  While  the  true 
ferments  or  enzymes  are  apparently  complex  chemical  sub- 
stances their  formation  is  due  in  every  case,  at  least  it  so  ap- 
pears, to  cell  action.  They  are  organic,  but  not  organized; 
yet  they  possess  many  of  the  properties  of  organized  bodies. 
On  the  other  hand  certain  finely  divided  metals,  especially  col- 
loidal platinum,  bring  about  a  number  of  reactions  which  were 
long  supposed  to  be  characteristic  of  the  true  ferments;  these 
reactions  are  further  modified  or  suspended  by  the  same  sub- 


ENZYMES    AND    OTHER    FERMENTS DIGESTION.  121 

stances  which  modify  or  suspend  the  ferment  actions  in  ques- 
tion. Based  on  this  behavior  it  has  been  attempted  to  relate 
the  true  ferment  action  to  the  "  catalytic  "  action  of  the  "  inor- 
ganic "  ferments.  All  the  ferments  seem  to  have  the  power 
of  decomposing  hydrogen  peroxide  in  quantity  or  catalytically, 
and  this  property  has  been  considered  as  perfectly  typical  or 
characteristic.  The  addition  of  a  number  of  mineral  sub- 
stances interferes  with  this  catalytic  decomposition;  in  this 
respect  the  action  of  prussic  acid  is  remarkably  energetic.  It 
has  been  found  that  a  minute  trace  of  colloidal  platinum  in 
dilute  solution  decomposes  a  greatly  excessive  amount  of  the 
peroxide,  and  further  that  extremely  dilute  prussic  acid  or 
corrosive  sublimate  checks  the  reaction  here  just  as  with  the 
true  ferment.  These  analogy  reactions  are  very  suggestive, 
even  if  they  do  not  explain. 

CLASSIFICATION  OF  THE  FERMENTS. 

With  our  present  knowledge  of  ferments  they  are  most  sat- 
isfactorily classified  according  to  the  character  of  the  decom- 
positions they  effect.  Two  distinct  kinds  of  action  are  easily 
recognized.  Many  ferment  changes  are  clearly  hydrolytic; 
that  is  the  reaction  follows  through  the  addition  of  a  molecule 
or  more  of  water  to  one  substance,  causing  it  to  break  up  into 
smaller  groups.  In  other  cases  the  reaction  appears  to  be  in 
the  nature  of  an  oxidation  process  in  which  the  ferment  causes 
or  brings  about  the  addition  of  oxygen  to  convert  one  sub- 
stance into  another.  Some  authors  limit  the  true  ferment 
reactions  to  changes  which  may  be  referred  to  one  or  the  other 
of  these  heads.  But  there  are  a  great  many  decompositions 
which,  while  they  may  not  be  so  clearly  defined  as  those  just 
mentioned,  must  still  be  looked  upon  as  of  ferment  origin. 
These  are  produced  by  bacteria,  and  in  all  probability  by  the 
enzymes  secreted  by  bacteria.  It  will  be  well  therefore  to  add 
a  third  general  group  to  make  the  classification  complete. 

KINDS  OF  FERMENT  REACTIONS. 

We   may   make    three    general    divisions    of    the    ferment 
changes,  as  follows : 


122  PHYSIOLOGICAL    CHEMISTRY. 

A.  Hydrolytic  Reactions. 

B.  Oxidation  Reactions. 

C.  Bacterial  Decompositions. 

A  brief  discussion  of  the  important  changes  coming  under 
each  one  of  these  heads  will  now  follow. 

A.     HYDROLYTIC  REACTIONS. 

The  most  important  of  our  ferment  reactions,  with  one 
exception  perhaps,  and  at  the  same  time  the  most  thoroughly 
studied,  the  changes  involving  hydrolysis  have  long  claimed 
the  attention  of  chemists.  The  true  nature  of  some  of  these 
reactions  is  easily  recognized  and  the  earlier  workers  in  this 
field  were  able  to  compare  the  behavior  of  the  enzymes  in 
question  with  that  of  dilute  hydrochloric  or  sulphuric  acid. 
In  other  very  important  cases  this  analogy  is  far  less  readily 
pointed  out  and  it  remained  for  recent  workers  to  satisfac- 
torily establish  the  true  relations.  When  malt  digests  starch 
or  when  certain  enzymes  convert  the  malt  sugar  formed  into 
glucose  the  general  nature  of  the  changes,  as  requiring  the 
addition  of  water,  may  be  shown  without  difficulty.  But  with 
the  behavior  of  pepsin  in  digesting  protein  we  have  more  dif- 
ficulty. Here  the  reaction  is  not  so  easily  followed,  and  the 
quantitative  relations  between  the  original  substances  and  the 
products  formed  are  more  complicated  than  is  the  case  with 
the  carbohydrate  decompositions.  However,  these  reactions 
hkewise  have  been  shown  to  involve  true  cases  of  water  addi- 
tion and  therefore  may  be  properly  grouped  with  the  carbo- 
hydrate reactions  as  hydrolytic. 

This  hydrolytic  ferment  activity  is  exhibited  mainly  in  the 
following  directions : 

1.  In  the  modification  of  carbohydrates  as  illustrated  by 
the  saccharification  of  starch  and  further  changes  in  the  sugar 
thus  formed,  and  in  other  sugars. 

2.  In  the  breaking  down  of  glucosides. 

3.  In  the  splitting  of  fats. 

4.  In  the  digestion  of  proteins. 


ENZYMES    AND    OTHER    FERMENTS ^DIGESTION.  I  23 

5.  In  the  so-called  fermentation  of  urea. 

Some  of  these  reactions  may  be  represented  by  definite  equa- 
tions. In  general  they  correspond  to  the  changes  produced 
in  the  same  substances  by  weak  acids  with  some  variations  in 
the  details.  The  salient  points  will  be  indicated  here,  leaving 
in  most  cases  the  fuller  discussion  for  following  chapters  which 
deal  with  the  details  of  digestion  phenomena. 

CHANGES  IN  CARBOHYDRATES. 

Amylase  or  Diastase.  Certain  enzymes  convert  starch 
paste  into  malt  sugar  by  a  reaction  which  is  indicated  by  the 
equation : 

(Ci.H.oOio)»  +  (H.0)„  =  (Ci2H..0u). 

The  enzymes  here  active  are  usually  described  as  diastases  or 
amylases,  the  terms  being  employed  in  the  plural,  since  the 
action  is  not  confined  to  a  single  substance.  Of  these  two 
terms  the  word  diastase  is  frequently  employed  in  the  broad 
sense  to  include  all  the  enzymes  which  act  on  the  starches  and 
sugars  formed  from  them,  while  the  term  amylase  is  employed 
to  describe  the  enzyme  which  changes  starch  intO'  malt  sugar. 
In  this  sense  it  will  be  used  here.  In  nature  ferments  of  this 
character  are  very  widely  distributed  and  serve  very  important 
functions.  They  are  active  in  the  changes  going  on  in  the 
vegetable  kingdom  during  the  growth  of  plants  and  the  ripen- 
ing of  fruits,  as  well  as  in  the  germination  of  seeds.  On  the 
commercial  scale  malt  represents  the  best  known  diastase- 
containing  substance.  In  the  animal  body  similar  substances 
are  found  in  the  saliva  and  in  the  pancreatic  secretion.  The 
first  of  these  is  called  salivary  diastase  or  ptyalin  and  the  sec- 
ond pancreatic  diastase  or  amylopsin. 

These  diastases  have  never  been  secured  in  anything  like 
pure  condition.  Very  active  solutions  which  digest  starch 
quickly  may  be  obtained  by  extracting  ground  malt  with  water, 
which  will  be  illustrated  later.  These  solutions  may  be  con- 
centrated at  a  moderate  temperature,  but  the  activity  of  the 
enzyme  is  destroyed  by  heat.     A  stronger  product  may  be 


124  PHYSIOLOGICAL    CHEMISTRY. 

secured  by  extracting  with  20  per  cent  alcohol  and  precipitat- 
ing the  solution  so  obtained  by  absolute  alcohol.  This  precipi- 
tate in  turn  may  be  redissolved  in  water  and  precipitated  again 
with  strong  alcohol  or  with  ammonium  sulphate,  to  secure  a 
purer  and  more  active  product. 

Besides  the  well-known  ferments  in  malt,  in  the  saliva  and 
in  the  pancreatic  secretion  the  follow^ing  may  be  mentioned 
here.  By  many  authors  the  active  substance  in  the  liver 
which  converts  glycogen  into  glucose  is  supposed  to  be  a  form 
of  diastase.  Others  hold  the  conversion  of  sugar  into  glyco- 
gen and  the  subsequent  and  gradual  formation  of  sugar  from 
glycogen  to  be  specific  vital  functions  performed  by  the  liver 
cells.  The  name  ccllitlasc  or  cytase  is  given  to  a  ferment  body 
which  is  found  in  many  vegetable  substances  and  which  has 
the  power  of  converting  cellulose  into  sugar.  Inidase  is  the  en- 
zyme which  acts  on  the  peculiar  starch  known  as  inulin  found 
in  many  vegetable  substances,  converting  it  into  fructose.  Inu- 
lase  does  not  appear  to  act  on  ordinary  starch  and  on  the  other 
hand,  malt  diastase  is  not  able  to  convert  inulin  into  sugar. 
Pcctinase  is  another  little  known  vegetable  enzyme  which  con- 
verts the  so-called  pectin  jelly  substances  into  a  reducing  sugar. 
The  original  pcctosc  in  the  seed  or  fruit  is  first  changed  into 
pectin  by  a  kind  of  coagulating  enzyme  called  pectase.  The 
true  behavior  of  these  bodies  is  not  yet  fully  known. 

Maltase  or  Glucase.  The  sugar  formed  by  amylase  from 
starch  is  known  as  maltose.  This  is  a  primary  product  and 
may  readily  be  further  converted  into  glucose  by  another  en- 
zyme occurring  in  malt  and  properly  known  as  maltase.  The 
nomenclature  of  these  enzymes  is  unfortunately  somewhat 
confused.  An  effort  has  been  made  to  name  them  systemat- 
ically, using  in  each  case  the  name  of  the  carbohydrate  or  other 
body  on  which  the  enzyme  acts,  as  the  first  part  of  the  descrip- 
tive term,  to  be  followed  by  the  suffix  ase.  Thus  amylase  re- 
fers to  the  enzyme  acting  on  amylose  or  starch  and  maltase 
to  the  enzyme  which  acts  on  maltose  or  malt  sugar.  But 
many  authors  do  not  follow  this  system  consistently;  hence 


ENZYMES    AND    OTHER    FERMENTS DIGESTION.  I  25 

we  have  as  describing  the  same  ferment  the  term  glucase  in 
use,  since  glucose  is  the  product  formed.  It  is  preferable  to 
employ  the  first  designation  or  rnaltase.  This  enzyme  belongs 
to  the  class  of  so-called  inverting  ferments  which  convert  di- 
saccharides  into  monosaccharides.  In  this  special  case  malt 
sugar  yields  glucose: 

C12H22O11  -L  H2O  =  2C6H12O6. 

This  maltase  is  found  not  only  in  malt  extract,  but  in  vari- 
ous yeasts  and  elsewhere  in  the  vegetable  kingdom.  It  is  also 
present  in  saliva,  but  in  small  amount,  in  the  pancreas,  the  liver 
and  in  the  blood.  The  formation  of  glucose  in  most  cases  is 
probably  a  secondary  reaction,  maltose  being  formed  first  as 
the  primary  product.  The  general  importance  of  this  reaction 
will  be  pointed  out  later,  as  it  plays  a  very  essential  part  in  the 
digestion  of  the  carbohydrate  foods. 

Lactase.  In  analogy  with  the  conversion  of  malt  sugar 
into  glucose  we  have  the  conversion  of  its  isomer,  lactose  or 
milk  sugar,  into  two  monosaccharide  groups.  This  is  accom- 
plished by  the  ferment  called  lactase  which  is  found  in  several 
kinds  of  yeast,  and  which  appears  to  be  distinct  from  the  mal- 
tase just  described.  The  change  of  milk  sugar  is  represented 
by  this  reaction : 

Q2Ho,0ii  +  H=0  —  CeHi^Oe  -h  CeHioOe. 
Glucose  Galactose 

Lactose  and  glucose  have  nearly  the  same  specific  rotation, 
[a]2)  =  52.5°  for  the  first  and  53°  for  the  second,  while  for 
the  galactose  it  is  about  83°.  The  inversion  may  be  readily 
followed  by  the  polariscope  therefore. 

As  to  the  distribution  of  this  enzyme  in  nature  there  is  still 
some  dispute.  According  to  some  authors  lactase  is  not  pres- 
ent in  the  gastric  juice  or  in  the  pancreatic  secretion,  but  other 
investigators  have  reported  finding  it  in  both  secretions.  It 
was  formerly  held  that  the  disappearance  of  milk  sugar  in  the 
body  is  due  largely  to  bacterial  actions,  as  some  of  these  organ- 
isms are  able  to  secrete  an  enzyme  which  acts  on  the  sugar. 


126  PHYSIOLOGICAL    CHEMISTRY. 

Invertase  or  Sucrase.  One  of  the  most  common  and  im- 
portant of  these  enzymic  reactions  is  the  inversion  of  cane 
sugar,  forming  glucose  and  fructose. 

C,=H=.On  +  H:0  =  CoH,=0»  +  CeH.^Oa. 
Glucose  Fructose 

The  name  invcrtin  or  invertase  has  been  given  to  the  enzyme 
which  accomphshes  this,  but  sucrase  would  be  in  better  accord 
with  the  general  nomenclature.  The  presence  of  this  invert- 
ing ferment  in  many  kinds  of  yeast  has  been  long  known. 
The  yeast  cell  alone  is  not  able  to  convert  cane  sugar  into  alco- 
hol and  carbon  dioxide;  an  inversion  must  first  be  brought 
about  in  some  manner.  In  old  yeast  or  in  yeast  in  which  the 
cell  has  been  destroyed  by  heat  or  by  mechanical  means  the 
inverting  enzyme  seems  to  be  present  in  greatest  abundance. 

Invertase  is  found  in  various  animal  secretions,  especially  in 
the  intestinal  juice.  The  inverting  power  of  this  secretion  is 
marked,  while  with  the  pancreatic  secretion  the  inverting 
power  is  much  less  pronounced.  In  the  gastric  juice  the  in- 
verting enzyme  is  said  to  be  present  in  some  amount  and  is 
sufficient  to  change  part  of  the  cane  sugar  of  the  food  inde- 
pendently of  the  acid  likewise  present. 

The  blood  does  not  appear  to  contain  this  invertase,  since  a 
solution  of  cane  sugar  injected  into  the  veins  is  eliminated 
later  by  the  kidneys  unchanged.  If  injected  into  the  portal 
vein,  and  thus  made  to  pass  the  liver,  inversion  takes  place 
rapidly,  as  that  organ  possesses  the  enzyme  in  quantity. 

Many  of  the  higher  as  well  as  the  lower  plant  organisms 
contain  invertase,  which  accounts  for  the  change  of  the  cane" 
sugar  into  invert  sugar  in  certain  cases.  In  general  this  reac- 
tion may  be  easily  followed  by  the  polariscope,  as  the  strong 
dextro-rotation  of  cane  sugar  gives  place  to  the  levo-rotation 
of  invert  sugar.  It  is  possible  to  make  a  fairly  pure  invertase 
solution  from  some  kinds  of  yeast,  and  such  a  solution  has 
certain  practical  applications  in  analytical  investigations.  By 
extracting  yeast  with  thymolized  water  a  solution  is  obtained 
which  rapidly  inverts  cane  sugar,  but  which  is  practically  with- 


ENZYMES    AND    OTHER    FERMENTS ^DIGESTION.  12/ 

out  action  on  malt  sugar  or  milk  sugar  and  which,  at  the  same 
time,  will  not  induce  alcoholic  fermentation.  This  property 
of  the  yeast  extract  is  made  use  of  in  the  determination  of  cane 
sugar  in  presence  of  the  others  just  mentioned. 

GLUCOSIDE   REACTIONS. 

For  our  purpose  it  will  not  be  necessary  to  go  into  many 
details  here.  A  few  decompositions  only  need  be  mentioned 
by  way  of  illustration.  The  glucosides  are  peculiar  com- 
pounds which  may  be  looked  upon  as  more  or  less  complex 
ethers  of  glucose.  They  are  decomposed  in  various  cleavage 
processes,  with  the  separation,  usually,  of  glucose  as  one  of  the 
constituent  products. 

Emulsin.  The  best  known  reaction  in  this  group  is  that 
which  takes  place  spontaneously  in  the  crushed  bitter  almond. 
Along  with  other  substances  this  kernel  contains  a  character- 
istic nitrogenous  glucoside  known  as  amygdalin  and  the  en- 
zyme called  emulsin.  In  presence  of  water  the  amygdalin 
breaks  up  in  this  way : 

GoH^rNOii  -f  2H2O  =  2C6H12O0  -f-  HCN  +  C6H5CHO, 

that  is,  glucose,  hydrocyanic  acid  and  benzoic  aldehyde  are 
formed.  In  the  uncrushed  dry  almond  this  reaction  does  not 
take  place  because  the  enzyme  and  glucoside  are  not  in  direct 
contact,  but  are  contained  in  different  cells.  The  same  result 
is  accomplished  by  distillation  of  the  bitter  almond  with  dilute 
acids. 

Similar  reactions  are  observed  with  salicin, 

GsHisOt  +  H.O  =  CeHi^Oe  +  CeH^.OH.CH.OH, 

Saliginin 

and  with  coniferin,  arbutin  and  other  glucosides.  A  related 
ferment,  known  as  myrosin,  converts  the  potassium  myronate 
found  in  black  mustard  into  allyl  mustard  oil,  CgHgNCS, 
glucose,  and  potassium  acid  sulphate. 


128  PHYSIOLOGICAL    CHEMISTRY. 

THE  SPLITTING  OF  FATS. 

The  general  reactions  of  fats  have  been  already  referred  to 
and  it  has  been  shown  that  in  general  they  may  be  broken  up 
by  the  action  of  water  in  the  form  of  superheated  steam : 
C,U,(C  n  H=„_,0=)3  +  3H=0  =  CsH^OsHa  +  3HC„H=,._,0.. 

The  action  of  the  pancreas  in  the  emulsification  of  fats  was 
recognized  as  early  as  1834  and  in  seeking  for  the  cause  of 
this  it  was  finally  found  to  depend  on  a  ferment  reaction,  and 
subsequent  soap  formation. 

Lipase  or  Steapsin.  The  active  principle  in  the  pancreas 
which  accomplishes  this  fat  splitting  was  first  called  steapsin 
and  afterwards  lipase.  The  details  of  its  behavior  in  the  di- 
gestion of  fats  will  be  explained  in  a  following  chapter.  Be- 
sides its  constant  occurrence  in  the  pancreas,  it  has  been  found 
in  the  blood,  the  liver  and  the  kidney.  More  recently  the 
existence  of  lipase  in  many  vegetable  substances  has  been  ob- 
served and  thoroughly  studied.  It  has  been  found  that  it 
hydrolyzes  some  of  the  simpler  ethereal  salts  very  readily  and 
on  this  behavior  is  based  a  method  of  recognition  of  conveni- 
ent application.  Of  these  ethereal  salts  ethyl  butyrate  is  pos- 
sibly the  best,  as  it  suffers  but  very  slight  change  by  the  action 
of  water  alone  at  ordinary  temperatures.  The  fat-splitting 
power,  or  enzyme  strength,  of  various  extracts  may  be  com- 
pared by  noting  the  amount  of  the  ethyl  butyrate  decomposed 
in  a  given  time  under  the  influence  of  the  extract.  The  extent 
of  hydrolysis  of  the  ethereal  salt  is  determined  by  titrating  the 
butyric  acid  liberated  w^ith  dilute  alkali. 

PROTEOLYTIC  REACTIONS. 

While  it  is  not  possible  to  write  equations  illustrating  accu- 
rately the  absorption  of  water  in  the  digestion  of  proteins,  as 
may  be  done  for  the  carbohydrates  and  the  fats,  yet  there 'is 
abundant  evidence  to  show  that  water  addition  is  in  most 
cases  the  characteristic  preliminary  change  here  also.  The 
action  of  superheated  steam  has  been  referred  to  in  a  former 
chapter,  but  at  the  ordinary  temperature  certain  proteolytic 
changes  take  place  which  are  the  results  of  enzyme  action.     At 


ENZYMES    AND    OTHER    FERMENTS DIGESTION.  I  29 

least  three  of  these  changes  have  been  thoroughly  studied,  and 
are  of  great  importance  in  the  digestion  of  foods. 

Rennet  or  Rennin.  It  has  long  been  known  that  a  certain 
product  found  in  the  stomachs  of  young  animals  and  especially 
in  the  calf's  stomach,  has  the  power  of  clotting  milk  rapidly, 
which  property  has  been  applied  in  the  manufacture  of  cheese. 
The  same  substance  is  found  also  in  the  pancreas,  and  the  same 
or  a  quite  similar  enzyme  in  a  number  of  plants.  In  fact,  this 
curdling  or  clotting  ferment,  like  others  already  described,  is 
quite  widely  distributed  in  nature.  As  occurring  in  the  stom- 
ach it  is  mixed  with  another  ferment,  which  will  be  described 
below,  known  as  pepsin.  The  two  substances  are  apparently 
quite  distinct  from  each  other  and  may  be  more  or  less  per- 
fectly separated.  Some  chemists  are,  however,  inclined  to 
consider  them  as  essentially  similar. 

Rennet  acts  on  the  protein  substance  casein,  throwing  it 
into  a  coagulated  or  clotted  form.  The  chemistry  of  the  reac- 
tion is  obscure  and  not  thoroughly  worked  out.  The  essen- 
tials of  what  is  known  about  it  will  be  given  later.  It  is  pos- 
sible to  obtain  an  active  extract  from  the  stomach  of  the  calf 
or  young  pig  which  may  be  kept  indefinitely  and  used  for 
cheese  making  or  other  purposes.  Formerly  in  the  cheese 
industry  small  fragments  of  the  dried  calf's  stomach,  preserved 
for  the  purpose,  were  mixed  with  the  milk  and  stirred  about 
to  induce  the  coagulation.  At  the  present  time  a  liquid  extract 
is  made  on  the  commercial  scale  by  the  action  of  an  appro- 
priate solvent  on  the  cleaned  stomach.  Glycerol  may  be  used, 
or  water  plus  a  small  amount  of  salicylic  acid  to  prevent  putre- 
faction. In  some  European  countries  certain  plants  have  been 
employed  in  the  place  of  animal  rennet  in  the  cheese  industry. 
Rennet  works  well  in  an  acid  medium  and  is  easily  destroyed 
by  alkalies. 

Pepsin.  The  best  known  and  most  thoroughly  studied  of 
the  proteolytic  enzymes  is  pepsin  which  has  the  power  of  di- 
gesting coagulated  albumin  in  an  acid  medium.  It  may  be 
obtained  best  from  the  mucous  membrane  of  the  hog's  stomach 


130  PHYSIOLOGICAL    CHEMISTRY. 

by  extraction  with  acidulated  water  or  glycerol.  In  the  stom- 
ach it  appears  to  exist  as  a  propepsin  or  zymogen,  in  which 
condition  it  is  known  as  pepsinogen.  The  action  of  acid  con- 
verts this  into  the  true  ferment.  Pepsin  is  very  sensitive  to 
the  action  of  alkalies  which  even  in  weak  solution  destroy  it 
or  materially  lessen  its  power  of  dissolving  protein.  In  pres- 
ence of  weak  acids,  preferably  hydrochloric  acid  of  o.i  to  0.2 
per  cent  strength,  it  forms  from  the  native  or  coagulated  pro- 
teins the  derived  products  known  as  albumoses  and  peptones. 
This  change  is  unquestionably  associated  with  the  addition  of 
a  number  of  molecules  of  water  to  the  original  protein  group. 

Commercial  "  pepsin  "  appears  in  commerce  in  the  form  of 
powder  or  scales.  The  latter  are  obtained  by  drying  the  ex- 
tract from  the  glands  of  the  stomach  on  glass  plates.  The 
product  is  far  from  pure,  as  it  contains  a  large  excess  of  other 
extractives.  Yet,  as  now  made,  one  part  by  weight  of  the 
scale  or  powder  is  capable  of  digesting  or  rendering  soluble 
two  to  four  thousand  parts  of  coagulated  albumin  in  the  form 
of  hard-boiled  eggs.  In  an  experimental  w-ay  products  of 
enormously  greater  activity  ha\"e  been  prepared;  it  is  said 
that  one  part  of  a  dry  pepsin  may  be  made  to  dissolve  three 
hundred  thousand  parts  of  coagulated  egg  albumin.  The  rel- 
ative strengths  of  pepsin  products  are  always  compared  by 
noting  the  amount  of  egg  albumin  or  washed  fibrin  which  they 
will  digest  in  an  acid  medium  of  definite  concentration. 

Pepsin,  like  most  of  the  enzymes,  is  precipitated  by  alcohol. 
In  aqueous  solution  with  a  little  acid  it  is  most  active  at  about 
40°  C,  and  loses  its  power  at  about  56°.  In  the  dry  condi- 
tion it  w^ith stands  perfectly  a  much  higher  temperature. 
While  hydrochloric  acid  is  usually  employed  as  an  aid  to  pep- 
sin digestion,  other  acids  may  be  used  with  equally  good  re- 
sults. Oxalic  acid,  lactic  acid  and  formic  acid  work  well,  but 
the  action  with  acetic  and  propionic  acids  is  weak.  In  presence 
of  alkalies  there  is  no  activity  and  certain  salts  also  interfere 
with  the  digestive  power. 

Through  fractional  precipitations  and  by  other  means  many 


ENZYMES    AND    OTHER    FERMENTS DIGESTION.  I3I 

attempts  have  been  made  to  obtain  a  "  pure  "  pepsin.  The 
strongest,  that  is  the  most  active,  products  so  secured  have 
been  analyzed.  The  results  do  not  differ  greatly  from  those 
found  on  analyzing  the  proteins,  yet  with  some  o-f  these  prod- 
ucts it  is  not  possible  to  obtain  the  ordinary  protein  reactions. 
We  have  no  clew  to  the  real  composition  of  the  substance. 
It  is  not  at  all  diffusible  through  parchment  and  must  have  a 
high  molecular  weight. 

It  is  stated  above  that  pepsin  and  rennin  are  possibly  iden- 
tical substances.  The  view  commonly  held  by  the  majority 
of  chemists  and  physiologists  has  been  that  they  are  distinct 
ferments  produced  possibly  by  different  regions  of  the  stom- 
ach, but  in  late  years  a  mass  of  evidence  has  been  accumulat- 
ing which  appears  to  throw  doubt  on  this  notion  and  suggest 
the  perfect  identity  of  the  two  proteolytic  enzymes.  The 
chemists  of  the  Pawlow  school  have  been  particularly  active 
in  advancing  this  theory.  According  to  them  an  extract 
which  shows  the  digesting  power  will  also  show  the  milk 
curdling  action.  If  it  is  strong  in  the  one  case  it  will  be  found 
strong  in  the  other  if  the  conditions  are  made  right.  This 
amounts  to  saying  that  the  same  enzyme  does  the  two  kinds 
of  work,  but  the  conditions  of  reaction,  concentration,  salt 
content,  etc.,  must  be  different  in  each  case.  A  commercial 
rennet,  for  example,  if  largely  diluted  with  0.2  per  cent  hydro- 
chloric acid,  will  show  a  strong  proteolytic  reaction,  while 
without  such  dilution  it  may  appear  quite  inactive.  It  is  held 
•further  that  the  milk  curdling  ferment  of  the  pancreatic  ex- 
tract is  probably  identical  with  the  trypsin  to  be  now  described. 

Trypsin.  One  of  the  most  active  and  important  of  the 
body  ferments  is  the  substance  which  occurs  in  the  pancreas 
and  known  as  trypsin.  It  may  be  extracted  from  the  minced 
organ  in  a  variety  of  ways  and  in  crude  form  is  a  commercial 
product.  In  its  action  it  bears  some  resemblance  to  pepsin, 
but  works  under  different  conditions.  While  pepsin  digests 
protein  compounds  in  dilute  acid  medium  trypsin  is  most  ac- 
tive in  presence  of  weak  alkali,  preferably  sodium  carbonate. 


13-  PHYSIOLOGICAL    CHEMISTRY. 

Action  may  be  observed  however  in  neutral  solution  and  even 
in  presence  of  a  trace  of  acid.  In  its  hydrolysis  of  proteins 
trypsin  goes  farther  than  pepsin.  The  action  of  the  latter, 
under  ordinary  conditions,  ends  with  the  production  of  albu- 
moses  and  peptones,  while  the  pancreatic  enzyme  carries  the 
splitting  process  to  the  extent  of  producing  a  number  of  com- 
paratively simple  amino  acids,  and  the  hexone  bases.  In  this 
respect  the  behavior  of  the  trypsin  is  comparable  with  that  of 
weak  sulphuric  acid  when  heated  with  the  protein  bodies.  As 
already  pointed  out  in  a  former  chapter  this  acid  resolves  the 
proteins  into  complexes  which  may  be  considered  as  the  con- 
stituent groups  of  the  large  molecule.  The  trypsin  digestion 
may  be  carried  far  enough  to  leave  products  which  fail  to  show 
more  than  a  very  faint  biuret  reaction.  This  reaction,  it  will 
be  remembered,  persists  as  long  as  anything  having  the  charac- 
teristics of  the  original  protein  substance  remains.  From  all 
this  it  is  evident  that  the  trypsin  is  a  hydrolyzing  agent  of 
marked  power. 

Of  the  real  nature  of  the  enzyme  nothing  is  known.  It  has 
never  been  isolated  in  a  condition  of  even  approximate  purity. 
The  pancreatic  extracts  of  the  market  contain  the  enzymes 
acting  on  fats  and  carbohydrates  as  well,  in  addition  to  a  very 
large  amount  of  other  matter.  The  active  ferment  is  very 
soluble  in  water  and  in  dilute  glycerol  or  dilute  alcohol,  but  in 
strong  alcohol  or  glycerol  it  is  insoluble.  In  presence  of  weak 
hydrochloric  acid  trypsin  is  quickly  digested  or  destroyed  by 
pepsin,  and  at  temperatures  much  above  50°  C.  it  soon  becomes 
inactive.  The  temperature  optimum  is  probably  about  40°  to 
45°,  in  weak  alkaline  solution,  but  the  statements  in  the  litera- 
ture on  this  point  are  somewhat  discrepant. 

THE  UREA  FERMENTATION. 

Urease  and  Other  Ferments.  Urine  exposed  to  the  air 
soon  becomes  alkaline  and  the  presence  of  ammonia  is  easily 
shown.  This  behavior  is  due  to  the  formation  of  ammonium 
carbonate  from  the  urea  by  a  reaction  which  may  be  expressed 
in  this  way : 


ENZYMES    AND    OTHER    FERMENTS DIGESTION.  133 

( NH=)  =C0  +  2H,0  =  ( NH4)  .CO3. 

The  agency  concerned  in  this  addition  of  water  molecules  was 
for  a  long  time  in  doubt,  but  investigation  finally  showed  it  to 
be  a  case  of  ferment  action.  In  all  urines  undergoing  this 
change  numerous  bacteria  are  present  and  by  separating  and 
making  pure  cultures  of  these,  several  species  have  been  found 
which  are  capable  of  decomposing  pure  solutions  of  urea. 
The  name  micrococcus  itrecc  has  been  given  to^  one  of  the  most 
active  of  these  bacterial  organisms.  It  has  been  found,  how- 
ever, that  the  action  is  certainly  due  to  a  soluble  product  or 
enzyme  secreted  by  the  bacteria,  since  it  may  be  brought  into 
solution.  This  solution,  after  the  most  careful  filtration  even, 
is  very  active  when  properly  made  and  will  quickly  induce  the 
ammoniacal  decomposition  in  urea  solutions. 

The  name  urease  has  been  given  to  this  soluble  ferment, 
which  must  belong  to  the  hydrolytic  group.  It  is  active  up 
to  about  50°  C.  and  is  much  more  stable  in  presence  of  alka- 
lies than  with  acids,  as  would  be  supposed  from  the  reaction 
it  produces.  The  enzyme  is  not  readily  extracted  from  the 
living  bacterial  cells;  these  must  first  be  destroyed  or  allowed 
to  die  out  in  process  of  making  strong  cultures.  The  cells 
holding  this  enzyme  are  very  widely  distributed  in  nature, 
being  found  in  the  air,  in  most  soils  and  in  river  water.  This 
accounts  for  the  usually  rapid  fermentation  of  urine. 

B.     OXIDATION  REACTIONS. 

Under  the  head  of  oxidation  reactions  it  is  very  easy  to 
include  some  in  which  the  essential  phenomenon  is  clearly  one 
of  addition  of  oxyg-en  to  the  decomposing-  substance.  This  is 
certainly  the  case  in  the  production  of  acetic  acid  from  weak 
alcohol.  In  other  cases,  however,  the  actual  nature  of  the 
chemical  change  which  occurs  is  more  obscure  and  the  classi- 
fication of  such  reactions  as  oxidation  reactions  is  possibly  open 
to  doubt,  as  will  appear  below. 


134  PHYSIOLOGICAL    CHEMISTRY. 

ALCOHOLIC  FERMENTATION. 

As  mentioned  at  the  outset  the  phenomena  of  alcoholic  fer- 
mentation were  the  first  to  claim  attention  and  many  of  the 
fundamental  conditions  were  empirically  established  long  be- 
fore the  part  played  by  yeast  in  the  process  was  recognized. 
After  the  investigations  of  Pasteur  our  knowledge  in  this  field 
rapidly  widened. 

Yeast.  The  common  agent  of  alcoholic  fermentation  is 
known  as  yeast,  but  under  this  term  are  included  a  very  large 
number  of  really  distinct  species.  In  fact  several  different 
genera  may  be  and  actually  are  employed  in  practice.  The 
following  table  gives  an  idea  of  the  relations  of  the  commoner 
organisms  classed  among  the  alcoholic  ferments.  The  yeasts 
with  many  other  cells  are  classed  in  a  group  of  the  budding 
fungi,  or  Eumycctcs,  as  distinguished  from  the  fission  fungi 
or  Schi::o}}iycctcs. 

Familx  Saccharomvcetes 

\ ^ 

Genus  Monospora       Saccharomyces       Schizosaccharomyces 


r  Cerevisiae 
Species  -|  EUipsoideus 

L  Pastorianus 
and  others. 

A  few  molds,  also,  bring  about  alcoholic  fermentation.  We 
have  included  here  Mucor  mucedo,  Mucor  racemosus,  Miicor 
Rouxii  and  others  which  are  not  in  any  way  technically 
useful. 

Ordinarily.  howe\er.  we  take  as  the  type  of  a  yeast  the 
common  beer  yeast  Saccharomyces  cerevisicc,  which  is  a  cul- 
tivated species  employed  in  fermentation  by  brewers  and  dis- 
tillers. In  the  natural  wine  fermentation  other  species  seem 
to  be  the  most  active.  These  are  found  on  the  skin  of  the 
grape  and  hence  find  their  way  into  the  juice  after  crushing. 
S.  apiculatus  and  6".  elUpsoideus  are  the  names  of  two  of  the 
most  important  of  the  species  active  in  this  way.     The  com- 


ENZYMES    AND    OTHER    FERMENTS DIGESTION.  I35 

men  beer  yeast  appears  in  the  form  of  nearly  spherical  cells 
having  a  diameter  of  8  to  9  /x.  It  is  active  through  a  com- 
paratively wide  range  of  temperature.  In  practice  the  fer- 
mentation of  malt  wort  to  produce  beer  is  carried  out  at  a 
very  low  temperature,  while  a  grain  mash  to  produce  common 
alcohol  or  whisky  is  fermented  at  a  high  temperature.  In  the 
one  case,  however,  weeks  are  required  to  complete  the  change, 
in  the  other  one  or  two  days. 

Like  most  similar  reactions  brought  about  by  living  cells  a 
limit  to  the  quantity  of  product  formed  is  soon  reached.  With 
ordinary  glucose  the  reaction  follows  approximately  according 

to  this  equation : 

CsHi^Os  =  2C2H6O  +  2CO2. 

As  the  alcohol  formed  accumulates  a  point  is  reached  where 
the  activity  of  the  yeast  cell  is  impeded  and  the  fermentation 
finally  stopped.  This  occurs  when  about  20  per  cent  of  alco- 
hol has  accumulated  as  a  reaction  product.  Very  strong  sugar 
solutions  do  not  ferment  at  all.  Indeed  some  of  the  common 
uses  of  cane  sugar  in  preserving  fruits  depend  on  this  fact. 
Some  of  the  conditions  of  fermentation  may  be  readily  illus- 
trated by  simple  experiments. 

Ex.  Make  a  strong  cane  sugar  syrup,  by  boiling  or  heating  together  10 
gm.  of  sugar  and  10  cc.  of  water.  Allow  to  cool  and  add  about  a  gram 
of  crumbled  compressed  yeast,  and  then  set  aside  for  several  days.  The 
solution  should  be  found  free  from  any  signs  of  fermentation. 

Ex.  Prepare  a  20  per  cent  solution  of  commercial  glucose  and  pour  50 
cc.  of  it  into  a  small  flask.  Add  some  yeast  and  allow  to  stand  two  days 
in  a  moderately  warm  place.  At  the  end  of  this  time  it  should  be  found 
in  active  fermentation,  as  shown  by  the  escape  of  gas  bubbles  and  the  odor 
of  alcohol.  If  allowed  to  stand  several  days  longer  in  the  ordinary  at- 
mosphere the  liquid  in  the  flask  usually  becomes  sour  from  acetic  fermen- 
tation. 

Ex.  Prepare  a  tube  with  sugar  solution  and  yeast  as  in  the  last  experi- 
ment. Close  it  loosely  with  a  plug  of  absorbent  cotton  and  heat  to  boil- 
ing, allowing  steam  to  escape  through  the  cotton.  If  the  tube  is  now 
left  to  itself  for  several  days  it  will  be  found  that  fermentation  has  not 
taken  place,  showing  that  heat  destroys  the  characteristic  property  of  the 
yeast  cell. 

Ex.  Prepare  another  tube  with  sugar  and  yeast  and  add  10  cc.  of  strong 
alcohol.     Shake  the  mixture   and   allow   to   stand.     No   fermentation   ap- 


136  PHYSIOLOGICAL    CHEMISTRY. 

pears,  as  the  activity  of  the  yeast  cell  is  destroyed  by  alcohol.  We  have 
good  familiar  illustrations  of  this  in  the  self-preservation  of  certain 
"heavy"  wines,  as  ports,  sherries  and  malagas,  while  "  light"  wines,  which 
contain  10  to  12  per  cent  of  alcohol  usually,  must  be  kept  tightly  bottled 
for  preservation. 

Ex.  Test  for  Alcohol.  We  have  many  tests  by  which  the  presence  or 
formation  of  alcohol  may  be  shown.  The  fermentation  of  a  saccharine 
liquid  is  followed  by  a  lowering  of  the  specific  gravity  as  may  be  easily 
found  by  experiment,  and  a  practical  quantitative  test  is  based  on  this 
fact.  A  simple  chemical  test  for  the  presence  of  alcohol,  which  in  most 
cases  is  sufficient,  is  the  following:  Add  to  the  clear  liquid  to  be  examined 
a  few  small  crystals  of  iodine,  warm  to  about  60°  C,  and  then  add  enough 
sodium  hydroxide  or  sodium  carbonate  to  produce  a  colorless  solution. 
An  excess  of  the  alkali  must  not  be  used.  In  a  short  time  bright  yellow 
crystals  of  iodoform  precipitate,  easily  recognized  by  their  color  and  odor. 
Certain  other  liquids  give  the  same  test. 

Ordinar)^  yeast  contains  the  soluble  ferment  called  invertase. 
which  has  been  already  referred  to.  This  may  be  shown  by 
experiment,  as  follows : 

Ex.  Crush  some  yeast,  add  water  and  wash  by  decantation  or  on  a 
filter  thoroughly.  Then  rub  up  the  washed  yeast  with  some  water  in  a 
mortar  and  add  the  mixture  to  a  solution  of  pure  cane  sugar  which  has 
previously  been  treated  with  a  few  drops  of  a  strong  alcoholic  solution  of 
thj^mol.  50  cc.  of  a  5  per  cent  cane  sugar  solution  will  answer.  The 
thymol  prevents  the  action  of  the  yeast  cell  fermentation,  but  does  not 
prevent  the  action  of  the  invertase.  The  mixture  should  be  kept  about 
24  hours  at  a  temperature  of  40°  to  50°  C.  At  the  end  of  this  time  it  is 
filtered  and  the  filtrate  tested  for  invert  sugar  by  means  of  the  Fehling 
solution.  Ether  and  chloroform  are  sometimes  employed  in  place  of  the 
thymol ;  the  latter  must  be  removed  by  heating  before  making  the  Fehling 
test. 

Zymase.  It  has  been  intimated  already  that  the  activity 
of  yeast  as  an  alcoholic  ferment  is  due  to  the  presence  of  an 
enzyme.  This  fact,  long  suspected  and  much  debated,  was 
finally  demonstrated  by  E.  Buchner,  as  explained  above. 
Buchner  rubbed  the  yeast  with  fine,  sharp  sand  and  water  and 
then  subjected  the  mixture  to  great  pressure.  The  liquid 
pressed  out  was  carefully  filtered  and  was  found  to  be  as 
active  as  the  original  yeast.  The  enzyme  in  it  he  called  zy- 
mase. It  clings  tenaciously  to  the  yeast  cell,  hence  the  neces- 
sity of  destroying  the  structure  by  grinding  with  sand,  and 
employing  great  pressure. 


ENZYMES    AND    OTHER    FERMENTS DIGESTION.  13/ 

Zymase  is  not  a  very  stable  ferment  and  in  the  solution 
obtained  is  soon  destroyed  by  other  ferments  present.  The 
yeast  extract  may,  however,  be  concentrated  at  a  Ioav  tempera- 
ture and  obtained  in  dry  form  which  is  more  stable.  Extracts 
made  from  yeast  by  simple  treatment  with  water  may  contain 
invertase  but  no  zymase.  It  seems  probable  that  the  ferment 
is  not  confined  to  the  yeast  cell.  It  has  long  been  known  that 
many  overripe  fruits  produce  a  small  amount  of  alcohol,  even 
when  the  possibility  of  the  presence  of  yeast  cells  is  entirely 
absent.  This  formation  of  alcohol  was  finally  ascribed  to  the 
cell  activity  of  the  fruits  themselves,  but  since  the  work  of 
Buchner  it  seems  more  rational  to  refer  the  appearance  of  alco- 
hol to  the  presence  of  an  enzyme  in  the  ripe  fruit. 

It  should  also  be  said  that  sugar  may  be  made  to  yield  alco- 
hol by  a  much  simpler  process.  It  has  been  found  that  a 
sugar  solution  mixed  with  a  little  potassium  hydroxide  and 
placed  in  bright  sunlight  yields  some  alcohol  and  carbon  diox- 
ide. This  is  of  course  a  purely  chemical  decomposition,  and 
suggests  the  possibility  of  chemical  reactions  in  other  cases. 
The  old  notion  as  to  the  necessity  of  the  presence  of  living 
cells  to  break  down  the  sugar  is  thus  completely  disproved. 

ACETIC  FERMENTATION. 

In  this  a  true  oxidation  takes  place,  the  oxygen  of  the  air 
being  employed  to  convert  weak  alcohol  into  the  acid  accord- 
ing to  this  reaction : 

QHcO  +  02=  QH.O2  +  H.O. 

The  active  agent  concerned  in  the  fermentation  oxidation  is 
the  cell  found  in  "  mother  of  vinegar." 

Mother  of  Vinegar  is  an  old  name  given  to  the  slimy  scum 
or  sediment  which  forms  in  weak  alcoholic  liquids  that  turn 
sour,  in  wine  or  cider,  for  example.  Microscopic  examina- 
tion shows  this  substance  to  consist  of  minute  cells  which 
have  received  the  name  of  Micoderma  aceti;  more  recently  the 
name  Bacterium  aceti  has  been  given  to  the  plant  organism. 


138  PHYSIOLOGICAL    CHEMISTRY. 

Thus  far  it  has  not  been  found  possible  to  isolate  a  soluble 
enzyme  from  the  cell  ferment.  One  may  be  present,  but  at- 
tempts to  obtain  it  have  failed. 

Besides  this  Bacterium  aceti  several  other  vinegar  ferments 
are  known.  Most  of  them  float  in  the  air,  and  when  lodged 
in  a  weak  alcohol  containing  certain  mineral  substances  pro- 
duce a  fermentation  quickly.  A  dilute  aqueous  solution  of 
pure  alcohol  will  not  ferment  in  the  same  way;  the  presence 
of  various  salts  and  organic  matters  in  addition  is  necessary. 
An  experiment  may  be  made  to  illustrate  vinegar  or  acetic  acid 
fermentation. 

Ex.  If  available  a  fruit  juice,  freshly  expressed  and  left  in  contact  with 
the  skin,  should  be  allowed  to  undergo  alcoholic  fermentation.  Or,  a 
sugar  solution,  as  described  some  pages  back,  may  be  allowed  to  ferment. 
The  weak  alcoholic  liquid  obtained  in  the  case  of  the  fruit  juice  will  next 
turn  sour  from  the  production  of  acetic  acid  by  the  action  of  the  germs 
on  the  skin.  In  the  case  of  the  alcohol  from  the  sugar  it  may  be  neces- 
sary to  add  a  little  "  mother  of  vinegar  "  from  a  vinegar  factory  to  induce 
the  fermentation.  Presence  of  the  air  is  necessary  to  complete  the  change. 
The  acid  strength  of  the  product  may  be  finally  tested  by  means  of  a 
standard  alkali  solution  and  phenol-phthalein. 

THE  OXIDASE  ENZYMES. 
We  come  now  to  a  very  brief  consideration  of  an  obscure 
but  interesting  subject  about  which  our  knowledge  is  of  com- 
paratively recent  origin.  In  certain  vegetable  substances  re- 
actions occur  which  are  ascribed  to  the  presence  of  a  class  of 
oxidizing  enzymes  called  oxidases.  These  changes  are  illus- 
trated by  the  blackening  of  an  apple,  potato  or  beet  which  is 
cut  and  exposed  to  the  air.  The  cut  surfaces  soon  turn  dark. 
If  the  same  substances  are  thoroughly  heated  before  the  cut- 
ting the  color  change  does  not  follow.  Potato  or  apple  pulp 
speedily  darkens  in  the  air,  but  if  previously  cooked  the  nat- 
ural light  color  persists.  To  account  for  these  and  many  sim- 
ilar reactions  it  has  been  assumed  by  many  chemists  that  the 
fruits  or  vegetables  in  question  contain  an  oxygen-carrying 
enzyme  and  at  the  same  time  some  chemical  substance  on 
which  this  can  act  with  the  production  of  color,  the  oxygen 


ENZYMES    AND    OTHER    FERMENTS DIGESTION.  I  39 

necessary  for  the  change  being  taken  from  the  air.  The  ac- 
tion of  this  enzyme  or  oxidase  may  be  shown  in  other  ways, 
especially  by  the  use  of  hydroquinol  and  pyrogallol,  which 
substances  yield  very  dark  solutions  when  oxidized.  It  is 
simply  necessary  to  make  an  aqueous  extract  of  certain  veg- 
etables and  fruits  and  add  this  to  the  aqueous  solution  of  the 
hydroquinol  to  produce  the  dark  color.  Here  the  enzyme 
appears  to  be  active  enough  to  carry  oxygen  to  the  hydro- 
quinol. 

Laccase  and  Tyrosinase.  These  are  the  names  which 
have  been  given  to  two  of  these  oxidases.  The  first  was  orig- 
inally found  in  the  sap  of  the  Japanese  lac  tree,  which  when 
expressed  and  exposed  to  the  air  darkens  and  produces  the 
well-known  lacquer.  The  same  laccase  is  said  to  be  one  of 
the  agents  which  brings  about  the  darkening  in  many  other 
saps  and  juices.  Tyrosinase  acts  on  the  phenol  derivative 
tyrosine  which  is  found  in  traces  in  many  vegetables  and 
causes  its  oxidation. 

It  is  held  by  many  physiologists  that  similar  substances  are 
found  in  the  animal  body  and  are  there  responsible  for  the 
oxidations  going  on  continuously.  These  assumed  enzymes 
must  be  found  especially  in  the  tissues  where  the  food  elements 
are  being  rapidly  broken  down  by  the  oxygen  carried  along  in 
the  blood  stream.  Thus  far,  however,  no  satisfactory  proof 
has  been  advanced  to  show  the  existence  of  such  oxidizing 
enzymes.  The  various  extracts  made  from  certain  organs  and 
investigated  from  this  standpoint  have  been  found  to  be  either 
inactive  or  to  work  in  a  limited  direction  only.  Extracts  from 
the  liver  and  spleen  have  been  found  able  to  convert  xanthine 
and  hypoxanthine  into  uric  acid,  but  this  fact,  important  as  it 
may  be,  is  far  from  throwing  any  light  on  the  oxidation  of 
sugar  to  form  water  and  carbon  dioxide.  Of  these  animal 
oxidases  it  may  be  said  in  general  that  our  knowledge  is  not 
exact  enough  to  justify  a  very  long  discussion  in  an  elemen- 
tary manual. 


140  PHYSIOLOGICAL    CHEMISTRY. 

C.     BACTERIOLYTIC  PROCESSES. 

The  term  bacteriolytic  is  applied  to  such  fermentation- 
splitting  processes  as  may  be  carried  out  by  bacteria  without 
the  addition  of  oxygen.  In  the  acetic  acid  fermentation, 
which  is  likewise  a  bacterial  process,  the  presence  of  oxygen 
is  necessary,  but  there  are  several  somewhat  analogous  reac- 
tions in  which  oxygen  is  not  required  and  these  are  included 
in  the  present  group.  It  must  be  admitted  of  course  that  the 
division  is  a  perfectly  artificial  one  based  on  convenience  rather 
than  on  marked  differences  in  agents  or  products.  Some  of 
the  reactions  classed  here  have  long  been  described  as  fermen- 
tations and  have  been  studied  in  connection  with  the  other 
common  ferment  changes.  These  will  be  taken  up  first.  But 
we  have,  in  addition,  further  changes  which  are  certainly  of 
the  same  general  character  and  call  for  like  treatment. 

LACTIC  AND  BUTYRIC  FERMENTATIONS. 

Why  milk  turns  sour  spontaneously  in  warm  weather  is  an 
old  Cjuestion,  but  it  was  not  satisfactorily  answered  until  after 
the  time  of  Pasteur's  pioneer  labors.  Following  his  work  on 
the  yeasts  Pasteur  took  up  other  problems  of  fermentation  and 
pointed  out  the  general  nature  of  the  reaction  by  which  the 
sour  substance  present,  lactic  acid,  is  formed.  He  found  the 
production  of  lactic  acid  to  depend  on  the  ferment  activity  of 
certain  microorganisms,  which  have  later  been  more  fully 
described  by  bacteriologists. 

Lactic  Acid  Bacteria.  It  was  found  that  lactic  acid  is 
formed  from  the  simple  sugars  by  a  splitting  process  which 
for  a  long  time  was  illustrated  by  an  equation  supposed  to 
represent  the  facts  quantitatively  : 

CeHi.Oa  =  2GHa03. 

It  was  also  recognized  that  not  merely  one,  but  many  species 
of  bacteria  are  capable  of  decomposing  sugar  solutions  in  this 
way.  Of  these  the  form  known  as  Bacillus  acidi  lactici  has 
been  perhaps  the  most  thoroughly  studied;  it  appears  to  be 
always  present  in  milk  which  has  soured  spontaneously,  and 


ENZYMES    AND    OTHER    FERMENTS DIGESTION.  I4I 

can  be  found  in  the  air,  especially  of  pastures  or  coAvsheds. 
Many  soils  also  contain  the  organism.  In  no  case,  however, 
is  the  reaction  a  perfectly  sharp  one ;  along  with  the  lactic  acid 
other  products  are  formed,  acetic  acid,  alcohol,  formic  acid, 
carbon  dioxide  and  hydrogen  being  the  most  common.  In 
some  cases  the  proportion  of  lactic  acid  is  relatively  small. 
The  formation  of  lactic  acid  may  be  illustrated  by  a  labora- 
tory experiment. 

Ex.  To  100  cc.  of  20  per  cent  cane  sugar  solution  add  an  equal  volume 
of  aqueous  malt  extract  and  10  to  15  grams  of  precipitated  chalk.  Inocu- 
late this  mixture  with  a  culture  of  lactic  acid  bacteria  and  keep  at  a  tem- 
perature of  about  40°  C.  for  some  days.  The  chalk  is  necessary  to  take 
up  the  acid  as  fast  as  formed ;  without  it  the  fermentation  soon  ceases,,  as 
the  ferment  is  extremely  sensitive  to  the  action  of  free  acid.  The  mixture 
must  be  shaken  from  time  to  time.  As  the  fermentation  progresses  the 
slightly  soluble  calcium  lactate  begins  to  separate.  In  a  good  fermenta- 
tion enough  of  this  forms  to  fill  the  fermenting  vessel  with  a  mass  of 
crystals.  These  crystals  are  redissolved  in  hot  water,  and  the  solution 
filtered.  The  filtrate  on  concentration  deposits  crystals  of  calcium  lactate, 
Ca(C3H50s)2.5H20,  which  may  be  collected  and  dried  between  folds  of 
filter  paper.  The  free  lactic  acid  may  be  obtained  by  decomposing  the 
calcium  salt  with  sulphviric  acid  in  the  proper  amount  and  shaking  out 
with  repeated  small  portions  of  ether.  The  lactic  acid  dissolves  in  the 
ether  and  is  left  when  this  is  evaporated.  Zinc  oxide  may  be  employed 
in  place  of  calcium  carbonate  to  neutralize  during  the  fermentation.  In 
this  case  zinc  lactate  forms,  from  which  the  acid  may  be  separated  by  dis- 
solving the  crystals  in  hot  water  and  decomposing  the  solution  by  means 
of  hydrogen  sulphide. 

Several  pure  cultures  of  lactic  acid  bacteria  can  now  be  obtained  for 
technical  use.  For  the  rapid  production  of  the  acid  Lafar  recommends 
Bacillus  acidificans  longissimus. 

Pure  lactic  acid  as  prepared  by  fermentation  is  a  thickish 
liquid,  with  marked  acid  taste  and  but  slight  odor.  It  is 
optically  inactive,  but  may  be  resolved  into  active  components 
by  treatment  with  strychnine,  which  crystallizes  with  the  levo 
modification.  This  common  fermentation  acid  is  employed 
for  several  purposes  in  the  industries  and  is  now  compara- 
tively cheap  since  the  introduction  of  methods  of  fermentation 
with  pure  cultures. 

Lactic  acid  fermentations  are  concerned  in  many  common 
operations.     In  the  leavening  of  bread  along  with  yeast  fer- 


142  PHYSIOLOGICAL    CHEMISTRY. 

mentation  there  is  usually  a  bacterial  fermentation  with  pro- 
duction of  acid.  In  some  kinds  of  bread  this  is  extremely 
important.  In  the  preparation  of  sauerkraut  and  many  pick- 
les a  lactic  acid  fermentation  is  the  characteristic  feature. 
Several  well-known  beverages  produced  from  milk  are  fer- 
mented in  such  a  manner  that  they  contain  lactic  acid:  kephir 
and  kumyss  are  illustrations.  Yeasts  and  the  lactic  acid  bac- 
teria work  together  in  many  instances  and  symbiotic  products 
are  the  rule,  perhaps,  rather  than  the  exception  in  fermenta- 
tions. In  the  milk,  industries  these  mixed  fermentations  are 
apparently  essential  in  the  ripening  processes,  and  in  certain 
distillery  fermentations  with  yeast  a  lactic  acid  fermentation 
is  encouraged  to  prevent  the  growth  and  action  of  the  bacteria. 
This  fermentation  lactic  acid  is  found  also  in  the  stomach  and 
the  intestine.  In  the  stomach  the  formation  of  any  large 
amount  is  usually  impossible  because  of  the  presence  of  hydro- 
chloric acid.  About  o.i  per  cent  of  free  hydrochloric  acid  is 
sufficient  to  impede  the  lactic  fermentation.  Free  mineral 
acids  are  not  present  in  the  intestine ;  the  organic  fermentation 
acids  may  therefore  be  formed  in  appreciable  quantities. 
Fermentation  lactic  acid  must  not  be  confounded  with  the 
isomeric  sarcolactic  acid  found  in  the  muscles. 

Butyric  Acid  Fermentations.  Another  very  important 
kind  of  acid  fermentation  is  that  which  results  in  the  forma- 
tion of  normal  butyric  acid : 

GH„0«  =  2Hc  +  2C0=  +  CHsOi. 

As  in  the  case  of  lactic  acid  this  butyric  acid  fermentation  is 
not  the  result  of  the  action  of  one  organism  only,  but  it  may 
be  produced  by  several,  and  furthermore  several  by-products 
are  always  produced  in  quantity.  The  above  reaction  is  then 
merely  a  limit  reaction,  which  is  approached  but  never  abso- 
lutely realized. 

In  the  milk  fermentation  the  lactic  acid  or  calcium  lactate 
formed  may  be  further  changed  to  butyric  acid,  the  necessary 
ferment  entering  from  the  air.  Most  river  waters  contain 
butyric  acid  bacteria,  which  bring  about  the  characteristic  fer- 


ENZYMES    AND    OTHER    FERMENTS DIGESTION.  1 43 

mentations  when  the  water  is  mixed  with  some  sterilized  milk, 
as  in  one  of  the  common  tests  carried  out  in  the  sanitary  ex- 
amination of  water.  Garden  soils  are  also  rich  in  some  of 
these  butyric  acid-producing  organisms,  and  may  be  used  in 
starting  a  fermentation,  as  may  be  illustrated  in  this  way : 

Ex.  Mix  100  cc.  of  a  5  per  cent  glucose  solution  with  four  or  five 
grams  of  fibrin  and  heat  to  boiling.  To  the  hot  solution  add  a  few  grams 
of  garden  loam  and  allow  the  liquid  to  cool  rapidly.  The  bacterial  spores 
resist  the  heat  while  other  forms  succumb  and  are  thus  disposed  of.  Keep 
the  mixture  at  a  temperature  of  about  37°  to  40°  C.  Fermentation  begins 
in  about  two  days  and  is  assisted  by  neutralizing  with  a  little  sodium 
hydroxide  from  time  to  time.  After  several  days  the  presence  of  butyric 
acid  may  be  shown  by  warming  some  of  the  liquid  with  sulphuric  acid, 
or  with  sulphuric  acid  and  alcohol.  In  the  latter  case  the  odor  of  ethyl 
butyrate  formed  is  very  characteristic. 

Butyric  acid  in  pure  condition  is  a  strongly  acid  liquid  pos- 
sessing a  rather  disagreeable  odor.  It  is  frequently  present 
in  the  stomach,  but  its  occurrence  there  is  really  abnormal. 
If  the  gastric  juice  contains  the  proper  amount  of  hydrochloric 
acid  a  butyric  acid  fermentation  is  not  possible.  With  dimin- 
ished hydrochloric  acid,  however,  bacterial  fermentations  can 
take  place.  In  the  arts  while  lactic  fermentation  is  desirable 
frequently,  and  encouraged,  the  butyric  fermentation  is  usually 
considered  very  objectionable  and  is  prevented  if  possible. 

Other  Fermentations.  It  will  not  be  necessary  to  explain 
at  length  any  other  cases  of  bacterial  fermentations,  as  these 
two  just  given  are  sufficient  for  illustration.  What  is  known 
as  the  mucous  fermentation  sometimes  takes  place  in  sacchar- 
ine liquids  or  in  wines  which  have  not  been  completely  fer- 
mented. A  slimy  mucilaginous  product  is  formed  here  which 
contains  a  kind  of  gum.  Certain  microorganisms  have  the 
power  of  decomposing  cellulose  and  the  operation  is  called  the 
cellulose  fermentation.  The  products  of  this  reaction  with 
certain  bacteria  are  mainly  gaseous,  hydrogen  and  marsh  gas 
predominating.  Certain  organisms  are  able  to  produce  fatty 
acids  also.  In  the  intestines  of  the  herbivora  changes  of  this 
character  take  place,  and  the  acids  produced  are  doubtless  of 
value  in  aiding  in  some  of  the  other  digestive  processes  which 
take  place  there. 


CHAPTER    VII. 

SALIVA  AND  SALIVARY  DIGESTION. 

It  has  already  been  said  that  the  sahva  contains  an  enzyme 
known  as  ptyalin,  the  function  of  which  is  to  begin  the  diges- 
tion of  starchy  foods.  It  remains  now  to  look  into  the  nature 
of  this  process  a  little  more  closely,  and  to  study  the  conditions 
of  this  kind  of  fermentation.  The  saliva  as  secreted  by  the 
three  large  pairs  of  glands  of  the  mouth  is  a  thin  liquid  with 
slightly  alkaline  reaction.  Because  of  the  constant  presence 
of  mucus  and  epithelial  cells  it  is  never  clear  but  presents  al- 
ways an  opalescent  appearance.  The  amount  secreted  daily 
varies  between  i  and  2  liters. 

In  the  older  literature  several  complete  analyses  of  saliva 
are  given,  but  less  importance  is  now  attached  to  these  than 
formerly,  since  a  great  degree  of  exactness  is  not  possible  in 
such  tests  and  besides  the  composition  of  the  secretion  cannot 
be  a  constant  one.  In  the  mean  the  amount  of  water  present 
is  99.5  per  cent.  In  the  0.5  per  cent  of  solids  about  0.2  per 
cent  consists  of  inorganic  salts  and  the  remainder  of  organic 
substances,  including  the  ferment.  Among  the  salts  there  is 
a  minute  trace  of  potassium  thiocyanate,  KSCN,  which  may 
frequently  be  recognized  by  the  test  with  ferric  chloride.  It 
is  not  known  that  this  substance  exerts  any  specific  function, 
and  in  different  individuals  it  is  present  in  different  amounts. 
Some  of  the  important  properties  of  saliva  may  be  illustrated 
by  simple  experiments. 

Ex.  After  washing  out  the  mouth  thoroughly  with  water  chew  a  piece 
of  rubber  or  other  neutral  insoluble  substance  to  stimulate  the  flow  of 
saliva.  Collect  25  to  50  cc.  in  a  clean  breaker  and  after  diluting  with  an 
equal  volume  of  distilled  water  allow  to  stand  a  short  time  to  settle.  Then 
filter  through  a  small  filter  paper  into  a  clean  vessel  and  use  the  filtrate 
for  the  following  tests  : 

To  a  few  cc.  of  the  clear  saliva  in  a  test-tube  add  several  drops  of  a 

144 


SALIVA    AND    SALIVARY    DIGESTION.  145 

dilute  solution  of  ferric  chloride.  This  gives  a  more  or  less  marked  red 
color  from  the  formation  of  ferric  thiocyanate.  A  very  strong  reaction 
must  not  be  expected.  Make  a  comparative  test  by  adding  a  like  amount 
of  ferric  chloride  to  dilute  solutions  of  potassium  thiocyanate. 

The  addition  of  solution  of  mercuric  chloride  discharges  the  color.  This 
test  is  of  value  in  distinguishing  between  a  thiocyanate  and  a  meconate, 
w^hich  sometimes  has  value  in  medico-legal  work. 

Test  the  reaction  of  saliva  with  neutral  litmus  paper.  It  will  be  found 
slightly  alkaline.  Now  add  two  or  three  drops  of  dilute  acetic  acid  and 
note  that  a  stringj'  precipitate  of  mucin  separates.  Filter  off  this  pre- 
cipitate and  test  the  filtrate  for  proteins  by  boiling  with  Millon's  reagent 
or  by  the  xanthoproteic  reaction. 

Make  a  thin  starch  paste,  about  a  gram  to  200  cc.  of  water,  and  observe 
that  it  does  not  respond  to  the  Fehling  sugar  test  already  described.  Mix 
ID  cc.  of  this  paste  with  5  cc.  of  the  filtered  saliva  and  warm  to  a  tem- 
perature not  above  40  cc.  for  about  15  minutes.  At  the  end  of  this  time 
apply  the  sugar  test  again.  A  yellow  or  red  precipitate  will  appear  now, 
showing  that  the  starch  has  been  converted,  in  part  at  least,  into  sugar. 

The  saliva  alone  fails  to  reduce  the  copper  solution,  as  should  be  shown 
by  trial. 

Pour  about  5  cc.  of  the  clear  saliva  into  a  test-tube  and  boil  a  few 
minutes ;  add  the  starch  paste  and  allow  to  stand  as  in  the  above  experi- 
ment. On  testing  with  the  copper  solution  no  sugar  will  be  found,  show- 
ing that  heat  destroys  the  activity  of  the  ferment. 

The  digesting  power  of  the  saliva  is  destroyed  also  by  the  addition  of 
a  small  amount  of  strong  acid  or  alkali  solution,  which  the  student  should 
prove  by  experiment. 

Saliva  is  practically  without  action  on  raw  starch,  as  may  be  shown  in 
this  way.  Stir  a  small  amount  of  uncooked  potato  starch  into  5  cc.  of 
saliva,  and  allow  to  stand  15  minutes  at  35°-40°,  and  filter.  Now  apply 
the  Fehling  test,  and  note  the  absence  of  precipitated  copper  suboxide. 

THE  CONVERSION  OF  STARCH. 

The  action  of  ptyalin  on  starch  is  a  compHcated  one  and  in 
all  details  cannot  be  satisfactorily  described.  In  many  re- 
spects the  digestive  behavior  of  the  enzymes  of  the  saliva  and 
of  malt  is  similar  to  that  of  weak  acid.  The  complex  insolu- 
ble starch  molecule  is  in  some  manner  broken  up  and  partly 
soluble  bodies  result.  This  change  is  at  first  unaccompanied 
by  hydration,  but  later  the  normal  enzymic  reaction  of  water 
addition  follows  and  the  dextrin  bodies  first  produced  become 
sugars.  Malt  sugar  is  formed  first,  and  in  the  case  of  acids 
this  gives  rise  finally  to  glucose  by  further  conversion.     But 


146  PHYSIOLOGICAL    CHEMISTRY. 

with  ptyalin  the  main  action  seems  to  end  with  the  produc- 
tion of  maltose;  at  all  events  no  large  amount  of  the  hexose 
sugar  is  formed.  A  little  maltase  is  said  to  be  present.  Fur- 
thermore the  whole  of  the  starch  is  not  brought  into  the  sugar 
condition;  a  portion  remains  in  the  form  of  a  dextrin.  In  an 
earlier  chapter  something  was  said  about  the  character  of  these 
dextrins. 

In  most  respects  the  behavior  of  ptyalin  is  very  similar  to 
that  of  malt  diastase,  which  can  be  shown  by  a  simple  experi- 
ment with  commercial  malt.  This  substance  is  usually  made 
by  germinating  barley  and  permitting  the  growth  to  continue 
some  days,  the  barley  in  moist  condition  being  spread  out  on 
a  so-called  malting  floor  to  encourage  the  growth  and  prevent 
overheating.  In  the  germination  the  enzyme  is  developed, 
probably  from  a  portion  of  the  protein  substance  present. 
When  the  action  has  gone  far  enough,  which  the  malster  rec- 
ognizes by  the  appearance  of  the  rootlet  thrown  out,  the  action 
is  checked  by  cjuick  drying,  leaving  the  diastase  in  permanent 
stable  condition.  This  malt  is  made  in  enormous  quantities 
for  use  in  breweries  and  distilleries.  In  the  germinating  seed 
in  the  ground  the  same  enzyme  is  formed  which  converts 
starch  into  soluble  food  for  the  young  plant. 

Ex.  Mix  about  10  gm.  of  pale  ground  malt  with  50  cc.  of  lukewarm 
water,  and  allow  the  mixture  to  stand  a  short  time,  with  frequent  stirring 
and  shaking.  Then  filter  and  add  the  clear,  bright  filtrate  to  a  thin  starch 
paste  made  of  10  grams  of  starch  with  250  cc.  of  water.  The  starch  paste 
must  be  cool  when  the  malt  extract  is  added.  Place  the  mixture  on  the 
water-bath  and  warm  to  5o°-6o°  C,  and  maintain  this  temperature.  Note 
that  the  liquid  gradually  becomes  thin  and  transparent.  From  time  to  time 
remove  a  few  drops  by  means  of  a  pipette,  and  test  with  iodine  solution. 
At  first  a  deep  blue  color  appears,  but  this  grows  weaker,  giving  place  to 
violet,  then  to  yellowish  brown  and  finally  no  color  is  obtained,  indicating 
completion  of  the  reaction.  The  starch  paste  is  first  converted  into  dex- 
trin and  finally  into  maltose.  Evaporate  the  solution  to  a  very  small 
volume  and  observe  the  taste  and  appearance  of  the  residue.  In  the  end 
product  'there  is  usually  about  80  per  cent  of  maltose  and  20  per  cent  of 
dextrin  when  made  at  the  temperature  of  this  experiment. 

It  has  been  found  in  practice  that  the  amount  of  malt  sugar 
formed  depends  on  the  temperature  and  duration  of  the  diges- 


SALIVA    AND    SALIVARY    DIGESTION.  147 

tion  with  diastase.  At  a  lower  temperature  with  longer  action 
the  conversion  of  the  dextrin  becomes  more  perfect.  This 
corresponds  with  the  behavior  of  the  pancreatic  diastase  which 
is  active  through  a  longer  period  usually  than  is  possible  with 
the  saliva. 

BEHAVIOR    OF    THE    DIASTASE. 

The  question  of  the  identity  of  the  malt  diastase  with  that 
from  saliva  is  still  a  disputed  one ;  while  some  writers  describe 
them  as  identical,  others  apparently  find  characteristic  points 
of  difference.  The  behavior  of  saliva  with  various  reagents 
has  been  pretty  thoroughly  studied ;  stronger  acids  and  alka- 
lies have,  of  course,  a  destructive  action,  but  experiments  seem 
to  show  that  very  weak  acids  favor  rather  than  retard  the 
digestive  power.  When  the  acid  strength  is  gradually  in- 
creased up  to  that  of  the  gastric  juice,  the  effect  of  the  ptyalin 
on  starch  paste  grows  weaker  and  finally  becomes  zero  long 
before  the  maximum  acidity  is  reached.  In  the  mouth  the 
action  of  the  saliva  is  certainly  largely  mechanical,  since  the 
time  for  any  other  action  is  entirely  too  short,  but  with  the 
passage  of  the  food  into  the  stomach  it  does  not  follow  that 
all  diastatic  digestion  ceases  because  of  the  acid  condition  of 
that  organ.  After  the  beginning  of  a  meal  some  time  is  re- 
quired for  the  commencement  of  hydrochloric  acid  secretion, 
and  a  further  time  before  enough  has  accumulated  to  seriously 
interfere  with  the  activity  of  the  diastase.  The  effect  of  the 
acid  is  dependent  on  its  concentration,  not  on  the  gross  amount 
present.  Up  to  a  concentration  of  about  o.oi  per  cent  the  acid 
seems  to  have  but  little  inhibiting  action.  Therefore  while 
this  amount  of  free  acid  is  accumulating  we  may  suppose  the 
salivary  digestion  to  go  on  in  the  stomach.  Later,  with  in- 
crease in  acid,  the  ptyalin  disappears,  possibly  through  gastric 
digestion. 

Many  salts  exert  an  influence  on  the  rate  of  diastatic  diges- 
tion. Usually  this  is  to  retard  the  action,  but  sodium  chloride 
and  other  neutral  salts  in  small  amount  have  a  beneficial  effect. 
With  other   substances  the  action   is   generally  unfavorable. 


I4S  PHYSIOLOGICAL    CHEMISTRY. 

Small  amounts  of  protein  matter,  or  preferably  the  syntonin 
or  acid  albumins  formed  from  the  proteins  by  combination 
with  traces  of  hydrochloric  acid,  seem  to  increase  slightly  the 
activity  of  the  salivary  diastase.  This  is  a  point  of  consider- 
able importance  in  explaining  possibly  the  continuation  of  the 
ptyalin  reaction  in  the  stomach.  Acid  combined  with  protein 
behaves  as  free  acid  toward  certain  indicators,  while  with  other 
indicators  it  does  not  show.  Starch  digestion  with  saliva  in 
a  mixture  containing  protein  and  hydrochloric  acid,  as  indi- 
cated by  dimethylaminoazobenzene,  cannot  continue,  but  if  the 
indication  is  merely  by  phenol-phthalein  the  ptyalin  action  may 
still  go  on,  since  in  this  case  the  acid  shown  may  possibly  be 
wholly  or  largely  combined  with  protein  substances.  Recent 
investigations  have  shown  that  under  such  conditions,  which 
are  probably  duplicated  in  the  stomach,  the  digestion  of  starch 
may  go  on  at  practically  the  normal  rate,  the  hydrochloric  acid 
being  rapidly  combined  with  protein,  and  therefore  compara- 
tively inert  with  ptyalin.  The  alkalinity  of  human  saliva  is 
usually  referred  to  as  due  to  the  presence  of  sodium  carbonate, 
but  soluble  phosphates  are  present  which  may  account  for  the 
reaction  as  shown  by  certain  indicators,  especially  by  litmus. 
With  phenol-phthalein  the  reaction  appears  neutral  ordinarily 
or  even  slightly  acid.  With  the  latter  substance  as  indicator 
it  is  generally  necessary  to  add  a  little  alkali  to  secure  neutral- 
ity. With  litmus  as  indicator  the  average  alkalinity,  expressed 
in  terms  of  NgoCO^,  is  0.15  per  cent.  This  reaction  seems  to 
vary  with  the  time  of  day  and  is  strongest  before  breakfast. 
Although  carbon  dioxide  is  present  in  saliva,  it  probably  occurs 
as  bicarbonate  rather  than  as  carbonate,  which  would  account 
for  the  reactions  noticed. 

j\Iany  soluble  substances  introduced  into  the  blood  in  any 
way  soon  appear  in  the  saliva.  This  may  be  shown  by  an 
experiment  which  illustrates  also  the  rapidity  of  absorption. 

Ex.  Swallow  2  grams  of  potassium  iodide  in  a  gelatin  capsule.  In  this 
manner  the  salt  is  gradually  dissolved  in  the  stomach  without  having  come 
in  direct  contact  with  the  mouth.     After  a  few  minutes  begin  testing  the 


SALIVA    AND    SALIVARY    DIGESTION.  1 49 

saliva  for  iodine.  At  first  the  tests  are  all  negative,  but  in  time  a  reaction 
appears  on  treating  the  saliva  with  something  to  liberate  the  iodine  in 
presence  of  starch  paste.  Solution  of  sodium  hypochlorite  may  be  used 
for  this  purpose.  The  time  required  to  exhibit  this  absorption  and  secre- 
tion with  the  saliva  varies  greatly  in  different  individuals. 


CHAPTER    VIII. 

THE  GASTRIC  JUICE  AND   CHANGES   IN  THE  STOMACH. 

The  gastric  juice  free  from  saliva  and  particles  of  food  is 
a  thin  liquid  with  specific  gravity  ranging  from  i.ooi  to  i.oio. 
It  contains  besides  certain  enzymes  some  small  amounts  of 
protein  matters,  a  little  sodium  chloride  and  traces  of  other 
salts  and  free  hydrochloric  acid.  Lactic  acid  is  also  frequently 
present  in  traces.  The  older  analyses  of  human  gastric  juice 
which  have  been  frequently  quoted  are  misleading,  as  they 
were  made  with  material  containing  saliva  and  food  products. 
By  aid  of  a  fistula  it  has  been  possible  to  obtain  a  fairly  nor- 
mal secretion  from  certain  animals,  especially  from  the  dog, 
and  much  of  our  knowledge  of  the  conditions  of  secretion  and 
variations  in  composition  has  been  secured  in  this  way.  The 
physiologically  important  substances  in  the  gastric  juice  are 
free  hydrochloric  acid,  pepsin  and  rennin. 

The  secretion  is  furnished  by  two  kinds  of  glands  known  as 
the  pyloric  glands  and  the  fundus  glands.  Both  groups  of 
cells  yield  the  two  enzymes,  but  the  pyloric  cells  do  not  seem 
to  furnish  an  acid  secretion.  It  is  probable  that  certain  of 
the  fundus  cells  only  are  concerned  with  the  acid  secretion. 
The  gastric  secretion  is  promoted  by  two  kinds  of  stimuli. 
Certain  chemical  substances  when  taken  into  the  stomach  have 
the  power  of  exciting  a  flow  of  the  juice  from  the  mucous 
membrane,  and  are  themselves  not  subject  to  gastric  digestion. 
Small  amounts  of  alcohol,  ether,  spices  and  meat  extracts  act 
in  this  way.  But  more  important  than  this  is  the  so-called 
"  psychic  "  stimulus,  depending  on  the  desire  for  food  and  the 
satisfaction  derived  from  partaking  of  it.  The  amount  of  the 
secretion  varies  with  the  nature  and  kind  of  food. 


GASTRIC    JUICE    AND    CHANGES    IN    STOMACH.  1 5  I 

THE   DIGESTIVE   AGENTS. 

Origin  of  the  Free  Hydrochloric  Acid.  The  material 
from  which  the  fundus  cells  produce  the  enzymes  and  the  acid 
is  the  blood.  But  this  is  always  slightly  alkaline  and  to  ac- 
count for  the  secretion  of  a  characteristic  acid  from  such  a 
source  has  long  been  a  puzzle  to  physiologists.  Several  hy- 
potheses have  been  advanced,  but  these  are  all  more  or  less 
faulty.  The  difficulty  is  not  with  the  liberation  of  hydro- 
chloric acid,  which  is  a  purely  chemical  question,  and  one 
which  may  now  be  explained,  but  with  its  secretion. 

The  blood  contains  always  a  small  amount  of  sodium  chlor- 
ide and  an  excess  of  carbonic  acid.  In  a  solution  containing 
these  two  things  some  double  decomposition  must  take  place 
with  production  of  a  little  free  hydrochloric  acid  and  sodium 
carbonate.  According  to  the  older  view  hydrochloric  acid  is 
so  much  stronger  than  carbonic  acid  that  the  liberation  of  the 
former  from  a  chloride  by  the  action  of  the  latter  is  impos- 
sible. But  this  view  leaves  out  of  consideration  the  effect  of 
a  much  greater  mass  of  the  weaker  acid  through  which  in  fact 
a  dissociation  of  the  chloride  is  to  a  certain  extent  accom- 
plished. But,  granting  this  kind  of  a  double  decomposition 
it  is  still  beyond  our  powers  to  explain  how  the  free  acid 
formed  in  the  cells  is  able  to  pass  in  one  direction  into  the 
stomach,  while  the  sodium  carbonate  produced  at  the  same 
time  wanders  in  the  other  direction  into  the  blood. 

This  acid  is  not  liberated  in  constant  amount  at  all  times 
but  its  flow  is  subject  to  the  influence  of  the  various  stimuli 
referred  to  above.  The  quantity  present  then  in  the  stomach 
may  vary  from  a  mere  trace,  or  zero  even,  to  a  maximum. 
This  maximum  may  be  0.2  or  0.3  per  cent  of  the  liquid  con- 
tents. How  it  is  measured  will  be  shown  below.  Just  what 
is  meant  by  the  term  free  hydrochloric  acid  will  be  presently 
explained. 

The  Enzymes.  In  an  earlier  chapter  the  general  nature 
and  behavior  of  the  two  gastric  ferments,  the  pepsin  and  ren- 


15-  PHYSIOLOGICAL    CHEMISTRY. 

nin,  was  pointed  out.  Whether  these  bodies  are  always  se- 
creted simultaneously  and  in  corresponding  amounts  is  not 
definitely  known,  but  that  this  is  the  case  is  often  assumed; 
it  will  be  recalled  that  the  followers  of  the  Pawlow  school 
consider  the  enzymes  identical,  as  referred  to  above.  In 
fact,  one  of  the  clinical  methods  in  use  for  the  estimation  of 
"  peptic  "  activity  is  based  on  the  measurement  of  the  rennet 
action  through  milk  coagidation.  The  process  seems,  how- 
ever, of  doubtful  value.  In  speaking  of  gastric  digestion  in 
adults  we  are  concerned  mainly  with  what  takes  place  through 
the  action  of  pepsin,  which  will  now  be  discussed. 

PEPTIC    DIGESTION. 

In  presence  of  free  acids  of  the  so-called  "  stronger  "  type 
pepsin  has  the  power  of  effecting  remarkable  changes  in  pro- 
tein substances,  which  have  been  the  subject  of  numerous  in- 
vestigations. In  the  stomach  hydrochloric  acid  only  comes 
into  play  and  it  first  gradually  converts  the  proteins  present 
into  acid-albumins  or  syntonin  bodies.  This  is  the  preliminary 
stage  in  the  digestion  of  these  food  substances  and  must  be 
accomplished  before  the  other  steps  in  the  stomach  are  pos- 
sible. 

In  this  reaction  the  hydrochloric  acid  enters  into  a  kind  of 
combination  with  the  protein.  The  product  has  just  been 
spoken  of  as  acid  albumin,  but  it  is  evidently  through  the  basic 
character  of  the  protein  complex  that  the  combination  can  take 
place.  The  protein  here  is  in  effect  a  very  weak  base.  The 
amount  of  acid  which  may  be  so  held  is  considerable,  and  may 
in  fact  amount  to  5  per  cent  or  more  of  the  weight  of  the 
protein.  With  certain  of  the  derived  protein  products  the 
weight  of  hydrochloric  acid  combined  is  even  larger,  at  times 
as  much  as  15  per  cent  of  the  protein  weight  being  so  held. 
These  derived  products  are  hydrolysis  products  with  smaller 
molecular  weight  and  evidently  more  available  hydroxyl  or 
amino  groups  to  hold  the  acid. 

It  is  generally  held,  as  just  stated,  that  this  acid  fixation  is 


GASTRIC    JUICE    AND    CHANGES    IN    STOMACH.  1 53 

the  first  step  in  the  gastric  digestion,  ahhough  some  authors 
claim  to  have  recognized  the  albumose  stage  as  the  primary 
one  in  some  cases.  While  this  acid  reaction  may  take  place 
in  pure  aqueous-acid  solution,  it  is  much  more  quickly  reached 
in  presence  of  pepsin,  as  is  the  case  in  the  stomach.  Experi- 
ments with  artificial  mixtures  show  that  the  combination  then 
is  almost  immediate,  as  is  made  evident  by  the  loss  of  "  free  " 
acidity,  to  be  explained  below.  Then  the  hydrolysis  goes  on 
and  the  various  derived  products  mentioned  in  a  former  chap- 
ter are  produced.  In  the  gastric  digestion  it  is  likely  that  the 
cleavage  does  not  usually  extend  beyond  the  production  of 
the  secondary  albumoses;  that  is,  not  much  real  peptone  is 
formed  in  the  time  naturally  consumed  in  normal  digestion. 
In  practice  the  larger  part  of  the  peptone  production  is  doubt- 
less left  for  the  trypsin  conversion. 

Hydrolytic  cleavage  beyond  the  acid  albumin  stage  is 
favored  by  abundance  of  free  acid,  but  in  absence  of  this  it 
still  goes  on.  In  actual  digestion  the  whole  of  the  hydro- 
chloric acid  may  be  combined  with  albumin,  leaving  some  of 
the  latter  in  excess  even,  yet  primary  and  secondary  albumoses 
will  appear  leaving  the  remaining  native  albumin  to  begin  the 
reaction  later.  In  other  words,  it  is  not  necessary  that  one 
stage  of  the  digestive  process  must  be  complete  before  the 
following  may  begin.  All  these  reactions  may  be  in  progress 
simultaneously,  and  if  needed  hydrochloric  acid  will  be  taken 
from  the  advanced  products  to  hasten  the  beginning  hydrolysis 
of  the  protein  yet  to  be  digested.  It  has  in  fact  been  shown 
that  hydrochloric  acid  in  combination  with  leucine  and  other 
amino  acids,  which  it  will  be  recalled  are  advanced  products  of 
proteolysis,  will  still  digest  fresh  albumin  rather  rapidly,  but 
not  as  well,  of  course,  as  the  equivalent  amount  of  free  acid. 

The  amount  of  acid  taken  up  by  an  original  native  protein 
substance  during  gastric  digestion  has  been  referred  to  al- 
ready. Starting  with  a  given  weight  of  pure  protein  hydro- 
chloric acid  may  be  added  until  a  distinct  reaction  is  shown  by 
dimethylaminoazobenzene.     This  indicator  behaves  as  a  very 


I  54  PHYSIOLOGICAL    CHEMISTRY. 

weak  base  and  will  show  no  free  acid  until  the  protein,  con- 
sidered as  a  basic  body,  is  saturated.  As  digestion  proceeds 
more  and  more  acid  must  be  added  to  complete  the  saturation. 
The  amount  of  acid  which  may  be  so  added  is  to  some  extent 
a  measure  of  the  advancing  cleavage.  With  phenol-phthalein, 
which  is  a  very  weak  acid,  the  whole  of  the  hydrochloric  acid 
behaves  as  "  free  "  acid.  The  acid  joined  to  the  protein  is 
"  combined  "  acid  as  far  as  the  dimethylaminoazobenzene  is 
concerned  and  this  indicator  may  be  used  to  show  the  excess 
of  free  acid  in  examinations  of  stomach  contents.  More  will 
be  said  about  this  below. 

As  hydrolytic  digestion  goes  on  the  amount  of  water  com- 
bined becomes  appreciable,  and  finally  may  reach  three  or  four 
per  cent,  as  has  been  determined  by  direct  experiment.  The 
analysis  of  the  albumose  and  peptone  products  shows  practi- 
cally the  same  thing;  these  substances  are  always  lower  in 
carbon  than  are  the  original  proteins  since  oxygen  and  hydro- 
gen have  been  taken  up  in  the  cleavage.  These  products  of 
diminished  molecular  weight  pass  from  the  stomach  in  the  con- 
dition of  hydrochloride  salts  into  the  small  intestine,  where 
they  undergo  a  new  order  of  changes. 

THE  ISOLATION  OF  PEPSIN. 

It  has  been  stated  already  that  not  one  of  the  enzymes  is 
known  in  e\'en  approximately  pure  condition.  Very  strong 
active  extracts  of  the  secretion  of  the  gastric  glands  of  ani- 
mals may  be  made  by  the  use  of  various  solvents.  Such  ex- 
tracts naturally  contain  much  besides  the  pepsin,  but  they  are 
suitable  for  experimental  and  other  purposes.  A  good  proc- 
ess originally  suggested  by  Wittich  is  illustrated  by  the  fol- 
lowing experiment : 

Ex.  Separate  the  fresh  mucous  membrane  of  the  hog's  stomach  from 
the  outer  coatings  and  mince  it  fine  in  a  meat  chopping  machine.  To  lO 
gm.  of  the  minced  membrane  add  200  cc.  of  glycerol  to  which  a  little 
hydrochloric  acid  has  been  added.  The  acid  should  amount  to  about  o.i 
per  cent  of  the  weight  of  the  glycerol,  and  may  be  added  in  the  form  of 
the  "  normal "  volumetric  acid  of  which  5  cc.  will  be  sufficient.     Allow  the 


GASTRIC    JUICE    AND    CHANGES    IN    STOMACH.  1 55 

mixture  to  stand  a  week  with  frequent  shaking,  then  filter  it  by  aid  of  the 
pump.  This  extract,  bottled,  will  keep  many  months.  For  use  5  cc.  of 
it  may  be  diluted  with  100  cc.  of  water  containing  the  right  amount  of 
hydrochloric  acid,  generally  o.i  to  0.3  per  cent. 

For  many  laboratory  experiments  a  fresh  aqueous  extract 
is  preferable  which  may  be  secured  in  this  manner : 

Ex.  The  washed  mucous  membrane  of  the  hog's  stomach  is  chopped 
fine  and  then  rubbed  up  in  a  mortar  with  sharp  sand  or  powdered  glass. 
Water  is  added  (plus  o.i  per  cent  HCl)  in  amount  ten  times  as  great  as 
the  weight  of  the  minced  membrane,  the  mixture  is  thoroughly  stirred, 
and  is  allowed  to  stand  over  night.  It  is  then  filtered  and  is  ready  for 
use.     Such  an  extract  is  relatively  strong. 

A  much  purer  product  may  be  secured  by  the  following 
process  as  worked  out  by  Kuehne  and  Chittenden : 

Ex.  Remove  the  mucous  membrane  of  a  hog's  stomach,  wash  it  thor- 
oughly with  water  and  spread  it  out  on  a  plate  of  glass.  Scrape  the 
membrane  with  a  knife  or  piece  of  glass  and  mix  the  scrapings  with  hydro- 
chloric acid  of  0.2  per  cent  strength.  About  half  the  membrane  should 
be  reduced  to  the  form  of  scrapings  and  for  this  mass  500  cc.  of  the  acid 
may  be  used.  Allow  this  to  digest  at  a  temperature  of  40°  C.  for  about 
two  weeks  in  order  to  convert  as  much  as  possible  of  the  protein  present 
into  peptone.  The  mixture  is  filtered,  and  to  the  filtrate  powdered  am- 
monium sulphate  is  added  to  complete  saturation.  The  object  of  this  is 
to  throw  down  the  pepsin  and  some  albumose,  the  peptone  formed  in  the 
digestion  being  left  in  solution.  This  precipitate  is  collected  on  a  filter, 
washed  with  saturated  ammonium  sulphate  solution  and  redissolved  in  a 
little  0.2  per  cent  hydrochloric  acid.  The  solution  so  obtained  is  placed 
in  a  tube  dialyzer  with  a  little  thymol  water  and  dialyzed  in  running  water 
until  the  sulphate  is  all  removed.  The  pepsin  solution  left  is  mixed  with 
an  equal  volume  of  0.4  per  cent  hydrochloric  acid  and  kept  at  40°  C.  5 
days  to  complete  peptonization  of  albumose  still  present.  Then  precipi- 
tation with  ammonium  sulphate  to  saturation  is  again  effected,  the  pre- 
cipitate collected  and  washed  as  before  and  taken  up  with  0.2  per  cent 
hydrochloric  acid.  This  solution  is  dialyzed  in  running  water  for  the 
removal  of  all  sulphate.  The  liquid  remaining  in  the  dialyzer  is  a  com- 
paratively pure  pepsin  solution.  It  may  be  concentrated  in  shallow  dishes 
or  on  glass  plates  at  a  temperature  not  above  40°  C,  and  leaves  finally 
a  scale  residue.  It  may  be  evaporated  perhaps  better  in  shallow  dishes 
placed  in  a  large  vacuum  desiccator  with  sulphuric  acid.  The  flakes  or 
scales  resulting  may  be  kept  in  dry  form  almost  indefinitely  and  will  be 
found  extremely  active. 

Commercial  Pepsin.  What  is  commonly  known  as  pepsin 
is  a  product  prepared  on  the  large  scale  from  the  hog's  stom- 


156  PHYSIOLOGICAL    CHEMISTRY. 

ach  and  preserved  in  dry  form.  Sometimes  the  mucous  mem- 
brane is  cut  into  shreds,  dried  at  a  low  temperature  and  ground 
to  a  powder,  in  wliich  condition  it  keeps  very  well.  In  pres- 
ence of  weak  hydrochloric  acid  this  powder  becomes  active 
and  is  able  to  digest  a  large  amount  of  albumin.  Usually, 
however,  the  mucous  membrane  is  extracted  in  some  manner 
as  illustrated  by  the  first  steps  described  in  the  Kuehne-Chit- 
tenden  process.  In  the  commercial  processes  the  following 
steps  are  much  simpler  however.  As  carried  out  in  the  United 
States,  they  aim  to  furnish  a  finished  dry  product,  one  part 
of  which  will  digest  3,000  parts  of  egg  albumin  prepared  in  a 
certain  way.  Several  different  methods  are  in  use  by  manu- 
facturers for  purifying  and  concentrating  the  extract  from 
the  stomach  glands. 

Some  Reactions  with  Pepsin.  The  behavior  of  peptic 
extracts  may  be  easily  shown  by  experiment.  For  this  pur- 
pose an  extract  made  by  the  use  of  glycerol,  as  described  above, 
is  very  convenient.  An  aqueous  extract  will  answer  if  freshly 
prepared. 

Ex.  Boil  an  egg  until  it  is  hard,  take  out  the  white  portion  and  rub 
it  through  a  clean  wire  sieve  with  fine  meshes,  by  means  of  a  spatula. 
Add  about  five  gm.  of  this  egg  to  100  cc.  of  0.2  per  cent  hydrochloric 
acid  in  a  flask,  and  then  add  2  cc.  of  the  glycerol  extract.  Keep  the  flask 
at  a  temperature  of  40°  C,  with  frequent  shaking.  In  time  the  egg  albu- 
min will  dissolve,  forming  an  opalescent  liquid.  Unless  the  flask  is  very 
frequently  shaken  the  solution  of  the  albumin  will  be  slow.  Use  the  solu- 
tion for  experiment  to  be  described. 

Ex.  To  2  cc.  of  the  glycerol  extract  in  a  test-tube  add  a  little  water 
and  boil  a  few  minutes.  Now  add  this  boiled  liquid  to  albumin  and  0.2 
per  cent  hydrochloric  acid,  as  in  the  last  experiment,  and  note  that  under 
the  same  conditions  digestion  does  not  take  place,  the  heating  having 
destroyed  the  active  enzyme.  In  like  manner  it  may  be  shown  that  the 
enzyme  is  destroyed  by  alkalies  or  stronger  acids. 

Ex.  Test  for  Albumose  and  Peptone.  About  an  hour  after  the  be- 
ginning of  the  digestion  explained  in  the  above  experiments  pour  off  about 
half  the  liquid,  neutralize  it  exactly  with  sodium  hydroxide  or  ammonia, 
and  then  saturate  with  powdered  sodium  sulphate  or  ammonium  sulphate. 
This  precipitates  albumose,  but  not  peptone.  Filter  off  the  precipitate  and 
apply  the  biuret  test  to  a  portion  of  the  filtrate,  using  only  a  very  small 
trace  of  copper  sulphate.  A  pink  color  should  be  observed.  Concentrate 
the  remainder  of  the  filtrate  from  the  albumose  precipitate  by  evaporation 


GASTRIC    JUICE    AND    CHANGES    IN    STOMACH.  1^7 

at  a  low  temperature,  pour  it  in  a  test-tube  and  add  some  strong  alcohol. 
This  gives  a  precipitate  of  peptone,  which  dissolves  by  adding  water.  The 
intensity  of  the  peptone  reaction  depends  on  the  duration  of  the  digestion. 
By  preparing  a  number  of  mixtures  and  keeping  them  at  a  temperature  of 
40°  for  different  periods  different  results  will  be  obtained.  It  is  an  in- 
structive exercise  to  test  samples  of  commercial  pepsin  in  this  way.  For 
such  tests  about  10  milligrams  should  be  used. 

In  practice  pepsin  is  always  valued  by  the  amount  of  protein 
matter  it  will  digest  in  a  given  time.  Hard-boiled  white  of 
egg  is  generally  employed  with  hydrochloric  acid  of  0.2  per 
cent  strength.  Sometimes  well-washed  fibrin  is  used,  with  a 
somewhat  weaker  acid.  As  an  illustration  of  practical  pepsin 
testing  the  following  may  be  given,  which  is  essentially  the 
process  of  the  U.  S.  Pharmacopoeia : 

VALUATION  OF  PEPSIN. 

Prepare  an  acid  solution  by  diluting  57.6  cc.  of  normal  volumetric  hydro- 
chloric acid  to  make  500  cc. 

In  50D  cc.  of  water  dissolve  0.0335  gn'^-  of  pepsin. 

Mix  50  cc.  of  the  acid  solution  with  50  cc.  of  the  pepsin  solution.  The 
resultant  100  cc.  will  contain  0.21  gm.  of  actual  hydrochloric  acid  and 
00033s  gin-  of  pepsin. 

Boil  a  fresh  hen's  tgg  fifteen  minutes,  then  cool  it  by  placing  in  cold 
water.  Separate  the  coagulated  white  part  and  rub  it  through  a  clean 
sieve  having  30  meshes  to  the  linear  inch.  Reject  the  first  portions  which 
pass  through.  Weigh  but  exactly  10  gm.  of  the  clean  disintegrated  sub- 
stance, place  it  in  a  200  cc.  flask  and  add  100  cc.  of  the  acid-pepsin  mix- 
ture last  described.  Put  the  flask  in  a  large  water-bath  or  thermostat 
kept  at  38°-40°  C.  and  let  it  remain  six  hours,  shaking  gently  every  fifteen 
minutes.  At  the  end  of  this  time  the  albumin  should  have  practically 
disappeared,  leaving  at  most  only  a  few  insoluble  flakes.  Much  depends 
on  keeping  the  temperature  constant,  and  shaking  at  regular  intervals. 
The  Pharmacopoeia  allows  a  latitude  of  2°,  but  in  exact  comparative  work 
it  is  better  to  keep  the  bath  exactly  constant  at  40°. 

In  the  above  test  if  the  albumin  is  all  digested  it  shows  that  the  pepsin 
has  a  converting  power  of  3,000  or  over,  which  meets  the  practical  require- 
ment of  the  Pharmacopoeia.  The  relative  digesting  power  of  stronger  or 
weaker  pepsin  may  be  ascertained  by  finding  through  repeated  trials  how 
much  of  a  pepsin  solution  mixed  with  the  acid  and  made  up  to  100  cc. 
will  be  required  to  dissolve  the  10  gm.  of  disintegrated  white  of  tg§ 
under  the  same  conditions.  The  process,  although  not  thoroughly  satis- 
factory, is  a  good  one  for  practical  purposes. 


158  PHYSIOLOGICAL    CHEMISTRY. 

THE  EXAMINATION  OF  STOMACH  CONTENTS. 

From  the  clinical  standpoint  the  examination  of  the  con- 
tents of  the  stomach  at  any  given  time  is  a  matter  of  consider- 
able importance.  The  examination  may  extend  to  the  detec- 
tion or  recognition  of  the  nature  of  various  solid  products 
present,  but  ordinarily  it  is  confined  to  the  detection  or  esti- 
mation of  the  acid  and  the  enzymes  on  which  the  functional 
activity  of  the  organ  depends.  For  such  examinations  it  is 
necessarv  to  collect  the  liquid  contents  of  the  stomach  by  the 
aid  of  some  kind  of  stomach  tube.  Vomited  matter  may  be 
used  for  the  same  tests.  In  any  event  it  is  preferable  to  have 
as  much  of  the  solid  contents  as  possible  along  with  the  liquid. 

Inasmuch  as  the  secretion  of  the  gastric  juice  does  not  take 
place  all  the  time,  as  was  pointed  out  above,  but  depends 
largely  on  the  action  of  certain  stimuli,  of  which  the  passage 
of  food  down  into  the  stomach  is  the  common  and  most  impor- 
tant one,  it  is  customar\^  to  encourage  the  flow  of  the  secre- 
tion by  giving  what  is  called  a  "  test  meal  "  some  time  before 
introducing  the  stomach  tube.  Unless  this  is  done  it  might 
be  possible  to  collect  from  the  stomach  a  liquid  practically 
free  from  either  acid  or  enzyme.  The  Ewald  test-meal  con- 
sists of  wheat  bread  and  water  or  tea  without  sugar;  50  gm. 
of  bread  to  400  cc.  of  water  is  an  average  meal.  The  content 
of  protein  in  this  would  amount  to  less  than  5  gm.  ordinarily, 
and  in  the  normal  stomach  enough  hydrochloric  acid  to  more 
than  combine  with  this  would  soon  be  secreted.  After  about 
an  hour  therefore  "  free  "  acid  should  be  detected  by  the  tests 
given  below.  With  a  meal  richer  in  proteins  more  time  would 
be  consumed  in  producing  an  excess  of  hydrochloric  acid.  In 
such  a  case  two  or  three  hours  might  elapse  before  it  would 
be  possible  to  detect  the  free  acid.  The  Riegel  test-meal  con- 
sists of  a  plate  of  broth  or  soup,  200  gm.  of  beefsteak,  50  gm. 
of  wheat  bread  and  200  cc.  of  water.  The  protein  in  this 
would  amount  to  about  60  gm.,  which  would  require  2  to  3 
gm.  of  hydrochloric  acid  for  preliminary  saturation.  Some 
hours  would  therefore  be  consumed  in  producing  this.     The 


GASTRIC    JUICE    AND    CHANGES    IN    STOMACH.  1 59 

detection  of  free  acid,  then,  in  such  a  case  would  be  evidence 
of  relatively  high  secreting  power. 

The  Detection  of  Free  Acid.  In  the  early  digestion  stages 
of  a  meal  rich  in  carbohydrates  organic  acids,  especially  lactic 
acid,  may  be  formed  by  bacterial  fermentation.  But  the 
amount  so  produced  is  usually  very  small  if  the  normal  secre- 
tion of  hydrochloric  acid  begins  in  the  proper  time.  The  or- 
ganic acids  produced  are  in  amounts  ordinarily  below  o.i  per 
cent.  Pathologically,  when  the  bacterial  fermentation  goes  on 
unchecked  by  the  production  of  hydrochloric  acid,  the  organic 
acid  may  accumulate  far  beyond  this  and  may  then  be  readily 
detected  by  the  processes  given  below.  At  present  the  detec- 
tion of  the  free  hydrochloric  acid  will  be  considered.  Some 
of  the  gastric  secretion  collected  by  a  pump  or  otherwise  is 
filtered,  and  the  filter  (always  a  small  one)  is  washed  with  a 
very  little  water.  The  mixed  filtrate  and  washings  is  used 
for  the  following  tests : 

DiMETH-iXAMiNOAZOBENZENE  Test.  To  a  few  cc.  of  the  gastric  filtrate 
add  a  drop  or  two  of  this  reagent  used  in  weak  alcohoHc  solution  (about 
0.2  per  cent).  Free  hydrochloric  acid  present  strikes  a  pink  or  even  red 
color  with  the  indicator.  Combined  acid  and  the  traces  of  organic  acids 
which  may  be  present  have  no  such  action. 

Congo  Red  Test.  This  substance  in  aqueous  solution  is  turned  blue  by 
very  dilute  hydrochloric  acid.  Organic  acids  do  not  give  the  test,  except 
when  present  in  relatively  much  stronger  solution. 

The  reaction  is  most  conveniently  carried  out  by  means  of  test  papers 
made  by  dipping  filter  paper  in  a  solution  of  the  coloring  matter  and  dry- 
ing. These  strips  are  dipped  in  the  gastric  filtrate  and  allowed  to  dry 
spontaneously. 

Methyl- Violet  Test.  A  dilute  violet-colored  aqueous  solution  of  this 
substance,  when  mixed  with  weak  hydrochloric  acid,  turns  blue.  The 
reaction  with  gastric  juice  is  faint,  but  when  care  is  observed,  characteris- 
tic. Organic  acids,  even  when  present  in  quantity,  do  not  give  the  test, 
which  was  first  successfully  used  for  the  detection  of  traces  of  mineral 
acids  in  vinegar.     Use  a  few  drops  with  2  cc.  of  the  gastric  filtrate. 

Guenzberg's  Reagent.  This  is  a  well-known  solution  and  is  made  as 
follows : 

Phloroglucin   2  grams 

Vanillin    i  gram 

'    Alcohol    100  cubic  centimeters 


l6o  PHYSIOLOGICAL    CHEMISTRY. 

To  make  the  test  for  free  hydrochloric  acid,  mix  5  cc.  of  this  solution 
with  5  cc.  of  the  gastric  filtrate  and  concentrate  in  a  glass  or  porcelain 
vessel  on  the  water-bath.  In  presence  of  free  hydrochloric  acid  the  liquid 
gradually  becomes  red  as  the  concentration  proceeds. 

Boas'  Re.\gext  for  Free  H\t)rochloric  Acid. 

Resorcinol    5  grams 

Cane  sugar   3  grams 

Alcohol,  so  per  cent   100  grams 

Add  a  few  drops  and  evaporate  as  above.  Color  appears  as  in  the 
other  test. 

Total  Hydrochloric  Acid.  By  the  use  of  the  above  tests 
the  excess  of  hydrochloric  acid  beyond  that  which  the  proteins 
and  bases  will  hold  is  recognized.  At  one  time  this  acid  was 
supposed  to  be  all  that  could  have  any  physiological  value. 
The  importance  of  that  combined  with  the  proteins  in  the  form 
of  acid  albumin  was  not  considered.  From  the  explanations 
given  above  it  is  evident  that  in  some  stages  of  the  digestive 
process  the  hydrochloric  acid  may  be  largely  or  wholly  in 
combination  and  therefore  not  in  a  form  to  be  recognized 
through  the  aid  of  the  tests  just  given.  From  experiments 
made  under  such  conditions  it  would  be  wrong  to  conclude 
that  the  stomach  is  secreting  no  acids.  It  has  been  found  that 
by  making  the  test  in  a  different  way,  employing  phenol- 
phthalein  instead  of  the  reagents  mentioned  above,  the  com- 
bined acid  may  be  readily  recognized.  To  do  this  we  must 
make  practically  a  quantitative  analysis,  and  the  method  em- 
ployed depends  on  the  proper  use  of  certain  indicators.  This 
will  be  taken  up  presently. 

The  Organic  Acids.  Under  normal  conditions,  as  already 
stated,  these  are  present  in  the  stomach  contents  in  very  small 
amounts  only.  As  their  formation  depends  on  bacterial  fer- 
mentation processes,  they  appear  only  when  hydrochloric  acid 
is  absent,  or  present  in  relatively  small  proportion.  Mineral 
acids  arrest  bacterial  fermentation  quickly,  from  which  it  fol- 
lows that  in  the  healthy  stomach  there  is  never  opportunity 
for  the  accumulation  of  much  lactic  or  other  acid  of  like  origin. 
These  acids  are  never  products  of  secretion  as  is  hydrochloric 


GASTRIC    JUICE    AND    CHANGES    IN    STOMACH.  l6l 

acid ;  they  are  not  formed  in  the  cells  of  the  walls  of  the  stom- 
ach, but  in  the  food  contents.  If  from  some  pathological 
cause  the  fundus  glands  fail  to  secrete  hydrochloric  acid  or 
secrete  it  in  traces  only,  then  the  fermentation  bacteria  can 
work  unhindered  on  the  carbohydrates  in  the  stomach  and 
produce  relatively  large  amounts  of  acid.  Lactic  acid  is  usu- 
ally the  most  abundant  of  these  fermentation  products,  but 
butyric  acid  is  occasionally  formed  and  also  acetic  acid. 

The  recognition  of  these  organic  acids  is  not  difficult  if  they 
are  alone  or  mixed  with  only  a  little  mineral  acid.  These  are 
of  course  the  cases  of  practical  importance;  much  hydrochloric 
acid  and  much  lactic  acid  could  not  be  found  together.  Among 
the  simpler  reactions  employed  the  following  with  iron  salts 
are  the  most  useful : 

Test  for  Lactic  Acid.  Prepare  a  dilute  solution  of  phenol  by  dissolv- 
ing I  gm.  of  the  pure  crystallized  product  in  75  cc.  of  water.  To  this  add 
5  drops  of  a  strong  solution  of  ferric  chloride,  which  produces  a  deep  blue 
color.  Five  cc.  of  this  mixture  suffices  for  a  test.  Add  to  it  a  few  drops 
of  the  liquid  containing  lactic  acid,  and  note  the  change  from  blue  to 
yellow.     (Uffelmann's  test.) 

A  weak,  colorless  solution  of  ferric  chloride  serves  also  as  a  test  sub- 
stance, as  its  color  becomes  much  deeper  by  addition  of  a  trace  of  lactic 
acid.     (Kelling's  test.) 

This  reaction  is  not  influenced  by  the  presence  of  small  amounts  of 
hydrochloric  acid,  as  can  be  readily  shown  by  adding  some  to  the  liquid 
to  be  tested.  The  color  change  depends  on  the  peculiar  behavior  of  ferric 
salts  with  organic  acids  in  general.  These  acids  are  relatively  weak  and 
with  ferric  iron  tend  to  form  "  undissociated "  salts  which  all  have  a 
deeper  color  than  have  those  with  the  stronger  acids. 

Both  of  these  tests  are  much  more  delicate  if  applied  to  the  product 
■obtained  by  shaking  out  the  gastric  juice  or  stomach  contents  with  ether. 
About  10  cc.  of  the  filtered  juice  may  be  shaken  with  100  cc.  of  ether 
in  a  separatory  ftumel  through  half  an  hour.  When  the  ether  is  drawn 
off  and  evaporated  slowly  the  lactic  acid,  if  present,  is  left  as  a  residue. 
This  residue  is  taken  up  with  a  few  cc.  of  water  and  used  for  the  tests. 

The  Amount  of  Acid.  It  has  just  been  shown  how  we 
are  able  to  recognize  the  free  acids  existing  in  the  gastric 
juice,  and  also,  under  certain  conditions,  that  in  combination 
with  the  protein.  An  equally  important  problem  is  the  deter- 
mination of  the  proportions  in  which  these  fractions  of  the 


1 62  PHYSIOLOGICAL    CHEMISTRY. 

total  acid  exi?t.  Several  different  schemes  have  been  pro- 
posed by  which  these  degrees  of  acidity  may  be  measured. 
The  total  acid  not  combined  in  the  form  of  inorganic  salts  may 
be  most  accurately  found  by  the  methods  of  ordinary  quanti- 
tative analysis.  The  total  chlorine  is  found  by  precipitation 
or  by  the  Volhard  titration.  The  total  bases  are  found  by 
the  usual  gravimetric  methods.  On  calculating  the  amount 
of  chlorine  necessary  to  combine  with  these  bases  an  excess  is 
left  over  which  must  be  considered  as  existing  in  the  form  of 
free  acid.  In  very  exact  work  the  traces  of  phosphates  and 
sulphates  present  must  be  also  determined  and  these  first  com- 
bined with  bases.  The  method  is  one  which  requires  great 
care  in  manipulation  and  besides  does  not  distinguish  between 
free  acid  and  that  held  as  acid  albumin,  and  this  is  a  very 
important  point. 

The  principle  of  another  general  method  may  be  illustrated 
in  this  way.  Three  portions  of  the  gastric  juice  of  5  cc.  each 
are  measured  off.  The  first  is  mixed  with  a  little  pure  sodium 
carbonate,  evaporated  and  ignited.  The  total  chlorine  is  so 
retained  and  may  be  found  by  the  \^olhard  titration.  The 
second  portion  is  evaporated  slowly  to  dryness  at  a  low  tem- 
perature, mixed  with  sodium  carbonate  and  ignited.  The 
chlorine  is  determined  in  the  residue.  This  represents  the 
fractions  combined  to  proteins  and  to  inorganic  bases,  as  the 
free  hydrochloric  acid  is  lost  in  the  original  evaporation  at 
low  temperature.  Finally  the  third  portion  is  evaporated  and 
ignited  without  any  addition.  The  chlorine  now  found  in  the 
residue  is  that  which  originally  existed  in  inorganic  combina- 
tion. AA'ith  these  three  operations,  as  is  at  once  apparent,  it 
is  possible  to  measure  the  element  in  the  three  kinds  of  com- 
bination. The  process  has  been  modified  and  improved  so  as 
to  be  fairly  exact. 

Attempts  are  now  made  to  determine  the  acid  accurately 
volumetrically  by  the  aid  of  indicators,  and  here,  it  may  be 
said,  if  we  can  neglect  the  lactic  acid  present,  pretty  good  re- 
sults are  possible.     But  if  the  lactic  acid  is  present  in  amount 


GASTRIC    JUICE    AND    CHANGES    IN    STOMACH.  1 63 

more  than  traces,  as  suggested  by  the  quahtative  tests  above, 
the  process  becomes  more  difficult.  Before  describing  the 
details  of  a  method  something  must  be  said  about  the  indica- 
tors themselves,  as  an  understanding  of  their  nature  and  be- 
havior is  necessary  for  much  that  is  to  follow. 

THEORY  OF  INDICATORS. 

The  indicators  employed  in  acidimetry  and  alkalinity  are  all  weak  acids 
or  weak  bases  themselves,  and  in  general  much  weaker  than  the  acids  or 
bases  in  the  determination  of  which  they  are  employed.  These  indicators, 
as  acids  or  bases,  form  salts  with  the  bases  or  acids  to  be  titrated;  it  is 
on  the  peculiar  properties  of  these  salts  that  the  value  of  the  indicators 
depends.  As  is  well  known  the  change  in  "  reaction "  in  employing  an 
indicator  is  accompanied  by  a  change  in  color.  This  change  in  color  is 
accounted  for  in  two  general  ways.  In  terms  of  the  dissociation  hypothe- 
sis the  indicators  themselves,  being  very  weak  acids  or  bases,  are  but 
slightly  dissociated  into  ions.  The  weak  acid  indicators  in  neutral  or  acid 
solution  exhibit  the  color  of  the  undissociated  substance.  Phenol-phthalein 
as  acid  or  undissociated  body  is  colorless.  Methyl  orange  in  neutral  or 
alkaline  solution  is  still  a  basic  body  and  is  yellow.  These  indicators  rep- 
resent two  distinct  types;  the  first  is  an  extremely  weak  acid  in  normal 
condition,  while  the  latter  or  its  related  body  dimethylaminoazobenzene  is 
a  very  weak  base.  All  the  other  common  indicators  may  be  ranged  be- 
tween these  two  in  properties  and  behavior.  Now  if  we  add  an  alkali  to 
phenol-phthalein  a  salt  is  formed.  For  example,  with  sodium  hydroxide 
the  sodium  salt  of  the  coloring  matter  is  produced  and  such  salts,  even 
with  the  weakest  acids,  are  dissociated  into  metallic  ion  and  colored  acid 
ion.  The  red  color  of  the  alkali  solution  of  phenol-phthalein  is  then  due 
to  the  liberated  red  ion.  The  undissociated  colorless  molecule  may  be 
represented  by  the  formula  OCOCeH^CCCGH^OH);,  while  the  acid  radical 
or  red  ion  may  be  given  the  formula  OCbC6H4C(C6H40H)C6H40.  With 
relatively  strong  alkalies  this  ion  is  always  set  free.  This  is  not  the  case 
with  weak  bases  or  very  weak  alkalies,  because  very  weak  bases  and  very 
weak  acids  do  not  form  stable  salts  with  each  other  in  aqueous  solution. 
Salts  formed  in  this  way  suffer  a  decomposition  by  water  which  is  called 
hydrolysis,  as  illustrated  by  this  equation : 

BA  -t-  H2O  =  BOH  +  HA. 

B  represents  a  weak  basic  radical  and  A  a  weak  acid  radical.  With  the 
weak  base  we  obtain  the  undissociated  HA  (weak  acid)  instead  of  the 
ion  A.  Phenol-phthalein  is  not  at  all  an  indicator  for  weak  bases  and  a 
very  poor  one  for  weak  alkalies  like  NH4OH.  A  considerable  excess  of 
such  bodies  must  be  used  to  overcome  the  hydrolyzing  power  of  the  water 
solvent. 

The  case  is  different  in  testing  for  or  measuring  weak  acids  by  aid  of 


164  PHYSIOLOGICAL    CHEMISTRY. 

phcnol-phthalein.  Assuming  it  in  solution  along  with  very  weak  acids 
(but  stronger  than  itself  of  course)  it  must  exist  in  the  practically  undis- 
sociated  colorless  condition.  If  we  are  measuring  acids  we  have  the  choice 
of  alkalies  and  may  therefore  use  one  of  suitable  strength,  for  example 
sodium  hydroxide.  This  added  to  the  mixture  neutralizes  the  stronger 
acid  first,  and  the  least  dissociated  or  weakest,  phenol-phthalein,  last. 
Even  the  very  weak  organic  acids  and  carbonic  acid  take  precedence  over 
the  indicator  in  this  respect.  Finally,  as  soon  as  the  last  trace  of  other 
acid  is  combined  and  the  first  sodium-phenol-phthalein  molecules  formed 
ionization  takes  place  and  color  appears.  Because  of  its  extreme  weak- 
ness as  an  acid  phenol-phthalein  may  be  used  in  this  way  in  the  titration 
of  a  wide  range  of  acid  substances.  It  may  be  used  in  titrating  the  acid 
combined  with  very  weak  bases  also,  for  example  the  SOi  in  alum,  as  the 
separated  base  has  no  effect  on  the  indicator. 

A1=(S04)3  -f  6NaOH  =  2AIO3H3  +  sNa.SO*. 

Quite  analogous  to  this  in  principle  is  the  titration  of  the  protein  com- 
bination of  hydrochloric  acid,  where  the  separated  protein,  like  the  alu- 
minum hydroxide  is  without  action  on  the  indicator. 

Prot.  HCl  +  NaOH  =  Prot.  -f  NaCl  +  H=0. 

This  reaction  will  be  studied  more  fully  later. 

Methyl  orange  and  several  related  bodies  are  employed  because  they 
exhibit  the  opposite  behavior.  Methyl  orange  is  represented  by  the  for- 
mula (CH3)2NCoH4N  :  N  —  CeHiSOsNa.  The  free  sulphonic  acid  is  known 
as  helianthin  and  has  the  formula  (CH3)2NCaH4N :  NGHiSOsH.  Its 
behavior  is  the;  same  as  that  of  the  methyl  orange.  Finally,  the  free  base, 
without  the  sulphonic  acid  group,  (CH3)2NC6H4N :  NCoHs,  is  now  largely 
used.  This  is  the  dimethylaminoazobenzene.  Its  behavior  shows  that  the 
HSO3  group  has  nothing  to  do  with  the  action  of  methyl  orange,  but  it 
is  not  yet  clear  on  what  group  the  basic  property  of  the  indicator  depends. 
Granting,  however,  its  behavior  as  a  weak  base  we  have  these  considera- 
tions to  notice : 

With  weak  acids  it  forms  extremely  unstable  salts  and  therefore  cannot 
be  used  in  the  titration  of  such  acids.  Carbonic  acid  is  practically  inert 
with  it.  But  bases,  even  very  weak  ones,  are  able  to  displace  it  from  its 
combinations  with  acids,  just  as  weak  acids  displace  phenol-phthalein. 
Weak  ammonia,  for  example,  which  combines  imperfectly  with  phenol- 
phthalein,  is  strong  enough  to  react  with  the  acid  combinations  of  the 
dimethylaminoazobenzene  or  methyl  orange.  Protein  in  the  so-called  acid 
albumin  combination,  in  which  the  protein  is  really  basic  in  character,  is 
stronger  than  the  indicator  and  able  to  displace  it  from  its  salts.  If  we 
add  a  weak  alkali  to  a  solution  of  the  red  salt  of  methyl  orange  the  color 
changes  immediately  on  the  neutralization  of  any  free  acid  which  may  be 
present.  The  yellow  color  of  the  undissociated  base  takes  the  place  of 
the  red  of  the  salt  or  free  ion.  With  the  very  weak  solutions  of  the 
indicator  used  the  merest  drop  of  alkali  should  be  sufficient  to  bring  about 


GASTRIC    JUICE    AXD    CHANGES    IN    STOMACH.  1 65 

the  change  in  the  indicator  salt  alone.  Assuming  in  solution  a  mixture 
of  free  hydrochloric  acid,  protein  and  hydrochloric  acid,  and  the  red 
methyl  orange-hydrochloric  acid  salt,  addition  of  weak  sodium  hydroxide 
would  produce  a  change  in  color  immediately  after  the  neutralization  of 
the  last  trace  of  free  hydrochloric  acid.  Any  excess  of  alkali  added  would 
separate  the  protein-acid  combination,  but  the  protein  would  behave  itself 
as  a  base  and  furnish  hydroxyl  ions  to  decrease  the  dissociation  of  the 
indicator  and  produce  the  characteristic  yellow.  Hence  the  "  neutral " 
point  is  reached  with  the  disappearance  of  the  actually  uncombined  HCl. 
With  a  weak  acid,  like  lactic  acid,  present  in  small  amount  the  condition 
would  be  practically  the  same.  Such  an  acid  is  but  slightly  ionized  and 
not  able  to  form  stable  salts  with  the  indicator. 

In  its  behavior  methyl  orange,  or  the  related  bodies,  is  not  quite  as  sen- 
sitive as  phenol-phthalein.  In  neutral  solution  it  is  slightly  dissociated, 
and  shows  therefore  a  mixed  shade,  due  to  the  undissociated  3'ellow  and 
the  red  basic  ion.  This  mixed  shade  is  a  drawback  in  titration,  as  the 
final  change  of  color  is  obscured  by  it.  In  employing  methyl  orange  it 
is  always  best  to  add  but  a  few  drops  of  the  weak  solution.  With  such 
a  trace  the  excess  of  acid  or  alkali  required  to  effect  ionization  or  the 
reverse  is  diminished. 

In  this  explanation  of  the  action  of  indicators  the  so-called  ionization 
theory  has  been  followed.  It  is  proper  to  say  that  according  to  a  later 
theory  which  is  finding  many  adherents,  the  color  changes  are  not  due  to 
any  differences  in  color  between  unionized  and  ionized  molecules,  but  to 
the  formation  of  dift'erent  forms  of  the  indicator  substances.  Phenol- 
phthalein  exists  according  to  this  notion  in  a  colorless  lactone  form  and 
as  a  red  carboxyl  acid.  The  action  of  alkalies  is  to  change  the  lactone 
ring  into  a  carboxylic  acid  group.  All  indicators  must  contain  a  so-called 
chromophoric  group  on  which  the  color  reaction  depends,  and  the  changes 
of  one  form  into  the  other  must  be  practically  instantaneous.  It  will  not 
be  necessary  for  our  purpose  to  go  into  the  details  of  this  theory,  although 
it  offers  a  very  simple  explanation  of  the  various  reactions  involved. 

Illustration.  Before  taking  up  the  actual  titration  of  the  stomach  con- 
tents a  practical  illustration  of  the  steps  may  be  found  useful.  To  this 
end  a  mixture  of  about  10  gm.  of  finely  divided  coagulated  egg  albumin 
with  ID  milligrams  of  powdered  pepsin  and  100  cc.  of  0.4  per  cent  hydro- 
chloric acid  should  be  made  up  to  200  cc.  with  water.  This  will  give  an 
acid  strength  at  the  very  outset  of  0.2  per  cent. 

Immediately  after  diluting  measure  out  three  portions  of  25  cc.  each  of 
the  thoroughly  shaken  mixture.  Filter  one  portion  (A)  at  once  and  wash 
the  residue  on  the  filter  with  water  several  times,  adding  the  washings 
to  the  filtrate.  Add  a  few  drops  of  phenol-phthalein  solution  and  titrate 
this  liquid  with  N/10  NaOH,  preferably  after  warming.  Warm  the  second 
portion  (B)  of  25  cc.  and  titrate  with  the  alkali  and  phenol-phthalein 
without  filtering.  In  general  the  result  here  will  be  higher  than  in  the 
first  case.  It  represents  the  total  acidity,  and  corresponds  to  one-eighth 
of  the  acid  taken.     In  the  titration  of  A  the  result  will  be  lower  because 


1 66  PHYSIOLOGICAL    CHEMISTRY. 

a  part  of  the  acid  combined  at  once  with  the  albumin  and  is  left  in  a  form 
not  yet  soluble.  To  the  third  portion  (C)  of  25  cc.  measured  off  add  2 
drops  of  the  weak  dimethylaminoazobenzene  indicator  and  titrate  directly 
with  the  N/io  alkali.  The  result  will  be  found  distinctly  lower  than  that 
with  A  or  B,  even  in  this  beginning  stage  of  the  process. 

The  remainder  of  the  albumin  and  acid  mixture  in  a  looseh'  stoppered 
flask  is  placed  in  a  water-bath  and  kept  as  exactly  as  possible  at  a  tem- 
perature of  40°  C.  through  five  or  six  hours.  The  mixture  is  shaken 
frequently  as  in  the  pepsin  test  described  above.  At  the  end  of  two  or 
three  hours  measure  out  two  portions  of  25  cc.  each ;  titrate  one  with 
phenol-phthalein  addition  and  the  other  with  dimethylaminoazobenzene. 
The  result  with  phenol-phthalein  present  should  be  exactly  the  same  as 
before,  while  with  the  other  indicator  it  will  probably  be  a  little  less  than 
in  the  first  case  and  not  much  more  than  half  the  acidity  shown  by  the 
phenol-phthalein.  After  the  digestion  has  continued  six  hours,  or  until 
practically  complete,  test  two  further  portions  of  25  cc.  each  in  the  same 
way.  The  total  acidity  as  measured  by  the  aid  of  phenol-phthalein  will  be 
found  constant,  while  with  the  dimethylaminoazobenzene  the  "  free  "  acid 
should  be  found  still  further  lowered  probably,  and  not  over  half  the  total 
acid.  The  exact  relation  of  the  free  to  the  total  acidity  depends  on  the 
strength  of  the  pepsin  and  the  amount  of  albumin  taken.  In  a  long- 
continued  artificial  digestion  or  in  presence  of  much  pepsin  the  acid  is 
gradually  combined  more  and  more  completely  because  the  basic  digestion 
products  formed  have  relatively  lower  molecular  w-eights  and  combine 
with  the  acid  more  or  less  perfectly.  Exact  numerical  relations  here  have 
not  yet  been  established  by  sufficiently  numerous  or  detailed  experiments. 

Titration  of  the  Gastric  Juice.  The  illustration  given 
above  shows  about  how  this  should  be  carried  out.  In  gen- 
eral as  large  a  volume  as  these  used  will  not  be  available,  but 
5  or  10  cc.  should  be  collected  by  the  tube  or  otherwise  for 
each  test.  In  testing  for  the  total  acidity  the  mixture  should 
not  be  filtered,  unless  the  digestion  is  far  advanced,  for  the 
reason  just  pointed  out.  A  part  of  the  hydrochloric  acid  may 
be  held  in  the  insoluble  residue.  In  testing  for  the  free  acid, 
however,  the  measured  portion  should  be  filtered  and  the  resi- 
due on  the  filter  washed  with  a  little  water.  The  whole  of  the 
free  acid  will  then  be  found  in  the  filtrate.  As  the  color 
change  with  the  indicator  here  used  is  not  as  sharp  as  in  the 
other  case  a  clearer  liquid  is  essential  for  the  test. 

These  two  titrations  give  us  the  total  acidity  and  the  free 
hydrochloric  acid,  but  do  not  measure  the  organic  acid  which 
may  possibly  be  present.     Attempts  have  been  made  to  esti- 


GASTRIC    JUICE    AND    CHANGES    IN    STOMACH.  1 6/ 

mate  this  by  aid  of  another  indicator.  Sodium  ahzarin  sul- 
phonate  has  been  used  for  this  purpose,  but  the  reaction  is  not 
as  sharp  as  desirable.  This  substance  appears  to  behave  as  a 
weak  acid,  but  one  not  as  weak  as  phenol-phthalein.  Lactic 
acid  may  be  titrated  with  it,  but  protein  separated  from  HCl 
behaves  as  a  base  toward  it.  Theoreticahy  the  three  indica- 
tors are  related  in  this  way,  as  illustrated  by  diagrams,  in 
which  H  Pht  represents  phenol-phthalein,  HAl  alizarin  sodium 
sulphonate,  Or  CI  the  hydrochloric  acid  salt  of  dimethylam- 
inoazobenzene  and  HL  lactic  acid : 

H  Pht        ^  HAl  1  Or  CI         1 

HClProt.  L  NaOH.  g^  P-^^^"  k  NaOH.  gCl  Prot  ^^NaOH. 
HCl  J  HCl  J  HCl  J 


It  has  been  shown  above  how  phenol-phthalein  and  the  methyl 
orange  bodies  act.  The  alizarin  sulphonate  as  standing  mid- 
way between  them  in  properties  is  influenced  by  the  protein 
which  may  be  separated  from  acid  albumin  in  titration  with 
NaOH.  Therefore  the  difference  between  the  titrations  in 
schemes  numbers  2  and  3  must  measure  the  lactic  or  similarly 
acting  organic  acid. 

Under  some  conditions  this  appears  to  be  true.  When 
there  is  relatively  much  lactic  acid  present  and  not  much  of 
the  digestion  products  a  fairly  good  end  reaction  is  obtained. 
This  corresponds  of  course  to  a  practical  case  and  the  indica- 
tor then  has  some  value.  But  as  digestion  goes  on  the  prod- 
ucts formed  are  more  or  less  basic.  While  not  strong  enough 
to  affect  the  phenol-phthalein  they  do  appear  to  act  on  the 
alizarin  compound  in  such  a  manner  as  to  diminish  the  alkali 
required  for  titration ;  the  free  hydrochloric  acid  is  thus  made 
to  appear  low.  It  is  plain  that  the  indicator  has  but  limited 
value. 

The  Amount  of  Pepsin.  Thus  far  the  detection  and  esti- 
mation of  the  acid  in  the  stomach  contents  has  alone  been  con- 
sidered, but  the  question  of  the  amount  of  pepsin  present  may 
be  of  equal  importance.     We  have  no  very  satisfactory  tests 


I  68  PHYSIOLOGICAL    CHEMISTRY. 

to  determine  this  amount.  l)iit  approximate  values  may  be  ob- 
tained by  observing  the  action  of  a  filtered  portion  of  the  gas- 
tric juice  on  some  albumin  solution  to  which  weak  hydro- 
chloric acid  has  been  added.  Comparative  tests  may  be  made 
in  this  manner : 

Prepare  some  egg  albumin  solution  of  about  2  per  cent  strength  (2  per 
cent  of  dry  albumin)  and  mix  this  with  0.4  per  cent  hydrochloric  acid  in 
equal  proportions ;  that  is  for  every  cubic  centimeter  of  the  albumin  solu- 
tion take  one  cubic  centimeter  of  the  acid  solution.  The  resultant  mix- 
ture has  an  acid  strength  of  0.2  per  cent  and  an  albumin  strengtli  of  i.o 
per  cent.  Measure  out  20  cc.  of  the  acid-albumin  mixture  and  add  to  it 
5  cc.  of  the  filtered  gastric  juice  or  stomach  contents.  To  another  20  cc. 
of  the  mixture  add  5  cc.  of  a  0.2  per  cent  pepsin  solution.  Incubate  both 
mixtures  through  24  hours  at  a  temperature  of  40°  C,  with  frequent 
shaking.  At  the  end  of  the  time  examine  both  the  incubated  liquids  for 
digestion  products.  To  this  end  neutralize  a  few  cc.  of  each  portion  with 
very  weak  alkali,  using  phenol-phthalein,  and  observe  whether  a  precipi- 
tate forms  or  not,  directly  or  on  warming  gently.  If  no  precipitate  forms 
in  either  fraction  the  digestion  has  gone  beyond  the  acid  albumin  stage, 
which  should  be  the  case  of  course  in  the  comparison  sample  with  the 
pepsin.  Next  test  5  cc.  portions  of  each  mixture  in  the  Esbach  albumin- 
ometer,  adding  the  usual  Esbach  reagent  (10  gm<  picric  acid  and  20  gm. 
citric  acid  with  water  to  i  liter).  This  reagent  precipitates  albumoses 
but  not  peptones,  when  used  in  excess,  and  from  the  extent  of  the  reac- 
tion in  the  tube  some  conclusion  can  be  drawn  as  to  degree  of  digestion. 
A  similar  test  should  be  made  -with  potassium  ferrocyanide  and  acetic  acid 
in  place  of  the  picric  and  citric  acids.  Ferrocyanhydric  acid  does  not  pre- 
cipitate peptones. 

In  another  general  method  the  action  on  solid  coagulated  protein  is 
observed.  White  of  egg  is  drawn  up  into  narrow  glass  tubes  having  an 
internal  diameter  of  about  2  mm.  and  coagulated  by  heat.  The  tubes  are 
then  cut  into  lengths  of  i  centimeter,  thus  exposing  the  ends  of  the  coagu- 
lated columns  of  albumin.  These  prepared  tubes  are  then  immersed  in 
the  filtered  gastric  juice  and  in  standard  pepsin  solution  to  be  compared 
and  kept  at  a  temperature  of  40°  some  hours.  The  change  in  length  of  the 
albumin  column  is  taken  as  the  measure  of  the  peptic  activity.  The  fil- 
tered gastric  juice  must  be  largely  diluted  with  water  before  making  the 
test,  as  salts  and  carbohydrates  present  interfere  with  the  normal  solution 
of  the  end  of  the  coagulated  mass.  The  amount  of  albumin  dissolved  un- 
der these  conditions  is  said  to  be  proportional  to  the  square  root  of  the 
ferment  strength,  but  the  rule  is  far  from  exact. 

It  has  been  explained  above  that  according  to  late  researches 
pepsin  and  rennin  are  believed  by  many  chemists  to  be  identi- 
cal substances.     As  the  milk  coagulating  behavior  seems  to  be 


GASTRIC    JUICE    AND    CHANGES    IN    STOMACH.  1 69 

much  more  easily  followed  and  measured  than  the  proteolytic, 
the  ferment  strength  is  frequently  determined  by  observing 
the  extent  of  the  coagulating  power.  The  test  may  be  made 
in  a  number  of  ways  and  has  already  found  clinical  application, 
but  the  real  value  of  the  process  remains  to  be  demonstrated. 

PRODUCTS  OF  PEPTIC  DIGESTION. 

Frequent  reference  has  already  been  made  to  the  question 
of  what  is  produced  from  the  food  proteins  during  peptic  di- 
gestion. In  answering  this  question  it  is  necessary  to  distin- 
guish between  what  may  be  formed  under  the  influence  of 
pepsin  and  hydrochloric  acid,  with  sufficiently  long  time  af- 
forded for  the  action,  and  what  actually  is  formed  in  the  few 
hours  in  which  food,  under  normal  conditions,  remains  in  the 
stomach.  On  this  subject  the  views  of  physiological  chemists 
have  undergone  various  and  marked  changes.  The  stomach 
has  long  been  considered,  popularly,  as  the  chief  organ  of 
digestion,  and  this  view  appeared  to  be  confirmed  by  the  results 
of  the  earlier  experiments  carried  out  with  artificial  digestive 
mixtures.  The  gradual  disappearance  of  coagulated  tgg  albu- 
min or  of  fibrin,  with  the  simultaneous  formation  of  soluble 
products,  is  a  phenomenon  easily  observed.  Various  precipi- 
tation reactions  served  to  recover  from  the  mixture  the  prod- 
ucts formed  and  these  were  early  spoken  of  as  "  peptones." 

At  this  time,  however,  the  distinction  between  real  peptones 
and  proteoses  was  not  thought  of.  It  remained  to  be  shown 
that  all  this  abundant  mass  of  material  formed  in  the  course  of 
a  few  hours'  digestion  consists  actually  in  the  main  of  prod- 
ucts preliminary  to  peptones.  This  later  knowledge  came 
somewhat  slowly  and  led  to  a  radical  view  of  just  the  opposite 
order  from  the  early  popular  one  of  the  function  and  impor- 
tance of  the  stomach.  If  the  stomach  is  not  the  principal 
organ  of  digestion,  it  was  asked,  what  is  its  real  value?  If 
the  operations  carried  out  there  may  be  accomplished  as  well 
later  in  the  intestine,  if  its  work  is  wholly  preliminary  and  if 
in  turn  these  preliminary  stages  are  not  really  essential,  what 
are  the  functions  for  which  the  presence  of  the  stomach  ap- 
pears to  be  "practically"  necessary?     A  number  of  remark- 


170  PHYSIOLOGICAL    CHEMISTRY. 

able  experiments  made  with  animals  threw  some  light  on  the 
question.  It  was  found  that  dogs  were  able  to  live  and  thrive 
without  the  stomach,  mixed  foods  of  various  kinds  being  al- 
most, if  not  quite,  perfectly  digested  in  the  intestine.  One  of 
these  dogs  was  kept  under  observation  several  years  after  com- 
plete removal  of  the  stomach,  and  in  other  cases  dogs  have 
been  fed  through  long  periods  by  direct  injection  of  food  into 
the  small  intestine,  the  connection  with  the  stomach  being 
meanwhile  completely  broken  by  ligature.  The  feces  of  these 
animals  were  found  to  be  practically  normal  in  most  cases. 

With  such  facts  in  view  a  school  of  chemists  following 
Bunge  have  come  to  the  conclusion  that  the  main  use  of  the 
stomach  is  in  the  destruction  of  bacteria  taken  in  with  the  food. 
The  acid  usually  present  in  the  gastric  juice  is  sufficiently 
strong  to  destroy  most  of  the  ferment  organisms,  which  if 
allowed  to  live  and  pass  into  the  alkaline  intestine  would  cer- 
tainly work  great  harm.  It  must  be  granted  that  this  view 
appears  plausible;  the  protection  of  the  intestine  through  the 
sterilizing  action  of  the  acid  is  beyond  question  of  prime  im- 
portance and  that  the  stomach  actually  accomplishes  this  end 
must  not  be  forgotten  in  any  discussion  of  the  relations  of  the 
one  organ  to  the  other.  It  is  well  known  what  happens  in  the 
human  stomach  when,  from  some  cause,  the  hydrochloric  acid 
is  temporarily  absent  or  greatly  diminished.  A  great  devel- 
opment of  organic  acid-producing  bacteria  follows,  and  the 
products  of  these  are  a  source  of  much  discomfort  without 
being  at  the  same  time  strong  enough  to  check  the  growth  of 
certain  pathogenic  bacteria. 

But  after  all  these  facts  have  been  given  due  weight  we 
must  still  admit  that  the  peptic  digestion  if  not  actually  "  essen- 
tial "  is  in  practice  really  important.  Some  peptone,  although 
not  a  large  amount,  is  formed  in  the  stomach  and  this  is  ready 
for  immediate  absorption  from  the  intestine.  The  proteoses 
are  doubtless  in  part  ready  for  absorption  also ;  the  final  diges- 
tion of  the  remainder  is  a  question  of  but  a  short  time  rela- 
tively, in  addition.     In  studying  the  products  of  pancreatic 


GASTRIC    JUICE    AND    CHANGES    IN    STOMACH.  I /I 

digestion  it  will  appear  that  some  of  them  are  identical  with 
those  formed  in  the  stomach  or  by  pepsin-hydrochloric  acid 
action  in  general.  Others  appear  at  first  sight  quite  distinct 
and  their  existence  leads  to  the  long-accepted  notion  that  the 
peptic  action  is  incapable  of  carrying  the  conversion  of  proteins 
through  to  the  final  stages.  The  conclusions  which  may  be 
drawn  from  the  most  recent  of  the  long  investigations  which 
have  been  carried  out  on  this  question  are  somewhat  conflict- 
ing, but  in  the  main  they  show  that  with  a  sufficiently  long 
time  allowed  the  end  products  of  peptic  and  tryptic  digestion 
are  essentially  the  same. 

Some  idea  of  the  extent  of  the  changes  taking  place  in 
reasonably  prolonged  peptic  digestion  may  be  obtained  from 
a  study  of  the  rapidity  of  combination  with  hydrochloric  acid 
which  has  been  already  referred  to  in  speaking  of  digestion 
experiments.  A  digestive  mixture  was  made  with  90  gm.  of 
coagulated  and  finely  divided  white  of  egg,  900  cc.  of  approxi- 
mately 0.2  per  cent  hydrochloric  acid  and  150  mg.  of  com- 
mercial pepsin.  Two  portions  of  this  mixture,  of  25  cc.  each, 
were  titrated  at  once,  one  with  use  of  phenol-phthalein  and 
the  other  with  dimethylaminoazobenzene.  The  remainder  of 
the  mixture  was  poured  into  a  large  flask  which  was  main- 
tained at  a  temperature  of  40°  in  a  thermostat  through  a 
number  of  days.  Titrations  were  made  from  time  to  time 
with  the  following  results,  25  cc.  of  the  mixture  being  always 
taken.  The  phenol-phthalein  titration  was  made  warm,  the 
other  cold. 


Time. 

Cc.  of  JVIzo 

NaOH  with 

Phenol-phthalein. 

Cc.  of  A^'io 
NaOH  with  Dimethyl- 
aminoazobenzene. 

at  once 

14 

9.0 

10  hours 

14-5 

8.5 

24       " 

14-5 

8.0' 

40       " 

14.7 

7-5 

96       " 

16.0 

6.9 

168       " 

16.6 

6.5 

It  appears,  therefore,  that  the  "  total  "  acidity  as  measured 
in  the  phenol-phthalein  titration  undergoes  a  slight  increase. 
The  hydrochloric  acid  remains,  and  added  to  it  are  some  diges- 
tive products  of  pseudo-acid  character,  but  strong  enough  to 


I/-  PHYSIOLOGICAL    CHEMISTRY. 

show  in  this  way.  On  the  other  haiifl  in  the  course  of  the 
week's  dig-estion  there  is  a  decrease  in  the  "  free  "  hydrochloric 
acid  as  measured  by  aid  of  the  dimethylaminoazobenzene  indi- 
cator. The  titration  here  is  not  as  sharp  as  with  phenol- 
phthalein.  but  close  enough  to  indicate  the  facts.  An  amount 
of  acid  corresponding-  to  9  cc.  of  the  N/10  alkali  was  "  free  " 
immediately  after  mixing.  About  5  cc.  had  evidently  com- 
bined with  the  egg-  albumin  to  form  the  acid  albumin.  As 
digestion  progressed  and  smaller  molecules  were  formed  more 
acid  was  required  to  unite  with  these.  Finally  the  perfectly 
uncombined  acid  amounted  to  the  equivalent  of  6.5  cc.  of 
alkali  only.  Before  the  end  of  the  digestion  bodies  were 
formed  which  probably  acted  as  both  acids  and  bases  with  the 
proper  indicators.     The  amino  acids  are  of  this  character. 

Results  of  somewhat  similar  nature  have  often  been  re- 
ported. The  short  table  below  contains  figures  given  by  Chit- 
tenden. A  mixture  was  made  containing  pure  egg  albumin, 
water,  pepsin  and  enough  0.2  per  cent  hydrochloric  acid  to 
produce  a  neutral  result  with  the  Giinzburg  reagent.  24  cc. 
of  the  acid  was  used  for  this.  The  mixture  was  placed  in 
the  thermostat  and  kept  warm  (38°)  for  some  days.  From 
time  to  time  a  test  was  made  for  free  acid  and  if  none  was 
found  more  was  added  to  reach  the  neutral  point  with  the 
reagent.  In  this  way  a  considerable  additional  amount  of 
acid  was  consumed.  The  table  gives  the  results,  which  show 
a  verv  marked  increase  in  the  amount  of  acid  combined. 


Time. 

Acid  Add< 

d  tc 

Show  Trace  of  Free  Aci 

2h.  45m. 

4-5 

cc. 

0.2 

per  cent  HCl 

5h.  30m. 

I.O 

2ih.  15m. 

30 

26h.  30m. 

1.0 

29h.  30m. 

1-5 

45h. 

1.0 

5lh. 

0.0 

69h. 

30 

94h. 

2.0 
TyTo 

The  irregularities  in  the  figures  are  doubtless  due  to  the  lack 
of  delicacv  in  the  indicator  used. 


CHAPTER    IX. 

THE  PRODUCTS   OF  PANCREATIC  DIGESTION. 

After  leaving  the  stomach  where  the  food  is  subjected  to 
the  influences  described  in  the  last  chapter  it  passes  into  the 
small  intestine,  where  it  comes  in  contact  with  other  agents 
of  change.  The  work  in  the  stomach  is  largely  preliminary 
and  serves  to  bring  the  food  into  a  finely  divided  homogeneous 
semi-liquid  condition,  in  which  it  may  be  readily  attacked  by 
the  new  digestive  enzymes.  As  explained,  the  chemical  actions 
in  the  stomach  are  comparatively  simple,  and,  leaving  out  of 
consideration  the  continuation  of  the  salivary  digestion,  are 
due  essentially  to  the  combined  effect  of  pepsin,  hydrochloric 
acid  and  protein  substances.  In  the  upper  part  of  the  small 
intestine,  however,  the  work  of  the  pancreatic  enzymes  is  much 
more  complicated;  at  least  three  kinds  of  reactions  take  place 
here,  due  to  the  three  distinct  types  of  ferments  in  the  pan- 
creatic secretion.  The  protein  digestion  begun  in  the  stomach 
is  completed,  the  carbohydrate  digestion  begun  by  the  saliva 
is  continued  or  completed,  while  the  fats,  not  yet  attacked,  are 
brought  into  a  condition  of  absorption  through  the  intestinal 
walls.  These  three  groups  of  changes  will  be  taken  up  in 
detail,  but  something  must  be  said  first  about  the  pancreatic 
juice  as  a  whole. 

COMPOSITION   OF   PANCREATIC  JUICE. 

For  obvious  reasons  it  was  not  possible  to  give  any  fair 
analysis  of  the  gastric  juice.  But  something  more  is  possible 
in  the  case  of  the  pancreatic  secretion  which  may  be  collected 
by  means  of  a  fistula.  Most  of  the  experiments  have  been 
made  with  dogs,  and  the  flows  collected  under  conditions  to 
give  a  secretion  as  nearly  normal  as  possible  show  that  it  con- 
tains approximately  in  the  mean  90  per  cent  of  water  and  10 

173 


1/4  PHYSIOLOGICAL    CHEMISTRY. 

per  cent  of  solids.  Of  the  solids  about  9  parts  are  organic 
and  I  part  inorganic.  The  organic  matters  are  largely  pro- 
tein in  character,  while  the  phosphates  and  carbonates  of  the 
alkali  metals  along  with  common  salt  are  the  important  min- 
eral constituents.  The  alkalinity  is  due  to  these  phosphates 
and  carbonates.  The  following  results  have  been  given  for 
the  composition  of  a  human  pancreatic  fluid  obtained  from  a 
fistula : 

Water    864.05  per  thousand. 

Organic  solids    132.51 

Inorganic   solids    3.44 

The  protein  amounted  to  92  parts  per  thousand. 

Among  the  organic  substances  we  have  the  enzymes,  traces 
of  fat,  soaps,  leucine  and  other  bodies  in  small  amount.  The 
specific  importance  of  these  substances,  aside  from  the  en- 
zymes, is  not  known. 

THE  BEHAVIOR  OF  TRYPSIN. 

In  an  earlier  chapter  a  few  words  were  said  about  the  func- 
tion of  this  important  pancreatic  enzyme  and  it  remains  to 
discuss  its  practical  relations  to  food  digestion.  The  acid 
chvme  from  the  stomach  passing  into  the  intestine  is  neutral- 
ized by  the  alkaline  pancreatic  fluid  and  the  bile.  In  this 
neutralized  condition  the  trypsin  is  able  to  continue  the  break- 
ing down  process  begun  by  the  pepsin,  and  the  proteoses 
formed  in  the  stomach  are  carried  further  to  the  peptone  stage 
and  made  ready  for  absorption.  From  what  was  said  in  the 
last  chapter  it  is  evident  that  the  trypsin  could  effect  the  pre- 
liminarv^  changes  also;  that  is,  it  is  not  really  necessary  that 
the  food  proteins  should  be  brought  into  the  proteose  condi- 
tion before  the  action  of  trv'psin  may  begin.  This  enzyme  is 
able  to  effect  the  complete  digestion  from  the  beginning,  and 
rather  rapidly  too,  which  may  be  illustrated  by  experiments, 
using  either  the  minced  gland  from  some  animal  or  an  extract 
made  by  the  aid  of  a  proper  solvent.     Such  an  active  extract 


PRODUCTS    OF    PANCREATIC    DIGESTION.  1/5 

may  be   secured   in   several   ways.     The   following  methods 
answer  very  well. 

DIGESTIVE   EXTRACTS. 

Ex.  Mince  a  hog's  pancreas  fine  and  weigh  out  about  lo  gxn.,  which 
cover  with  absolute  alcohol  in  a  small  bottle.  Cork  and  allow  to  stand 
over  night.  Then  pour  ofif  the  alcohol,  which  is  added  to  remove  water, 
and  squeeze  out  the  residue.  Return  to  the  bottle,  add  lo  cc.  of  glycerol 
and  allow  the  mixture  to  stand  about  a  week  with  frequent  shaking.  At 
the  end  of  this  time  pour  or  strain  off  the  glycerol  which  is  now  a  fairly 
strong  pancreatic  extract,  and  able  to  act  on  the  three  classes  of  food 
stuffs.  It  has  been  found  by  experience  that  the  extracts  from  beef  and 
hog  glands  are  not  quite  the  same  in  digestive  activity,  but  the  hog's  pan- 
creas yields  a  product  suitable  for  all  practical  purposes,  and  which  keeps 
a  long  time  when  made  in  this  manner. 

Ex.  An  active  pancreas  powder  which  keeps  indefinitely  is  also  very 
useful  and  may  be  made  in  this  way.  Remove  the  adhering  fat  as  care- 
fully as  possible  from  a  hog  or  beef  pancreas  and  mince  it  fine  in  a  meat 
chopping  mill.  The  disintegrated  substance  is  treated  as  above  with  an 
excess  of  absolute  alcohol  to  remove  water.  The  alcohol  is  poured  off 
and  the  residue  pressed  dry.  This  residue  is  mixed  with  ether,  allowed  to 
stand  an  hour,  and  then  freed  from  ether  by  pouring,  pressing  and  air 
evaporation.  This  treatment  removes  practically  all  the  water,  traces  of 
fat  remaining  and  other  substances  soluble  in  alcohol  and  ether.  What 
is  left  is  thoroughly  air  dried,  ground  to  a  fine  powder  and  sifted  through 
gauze  with  20  to  30  meshes  to  the  inch.  The  powder  so  secured  may  be 
kept  in  a  stoppered  bottle.  In  digestion  experiments  the  powder  may  be 
used  directly,  or  an  extract  may  be  employed.  This  is  best  obtained  by 
soaking  a  few  grams  of  the  powder  with  fifty  times  its  weight  of  thymol 
water  through  24  hours. 

Some  of  the  conditions  of  pancreatic  digestion  may  be  illus- 
trated by  very  simple  experiments. 

Ex.  Pour  25  cc.  of  a  I  per  cent  solution  of  sodium  carbonate  (crys- 
tallized salt)  into  each  of  several  small  flasks  or  test-tubes.  Add  to  each 
half  a  cc.  of  the  glycerol  extract  of  pancreas  and  about  a  gram  of  finely 
divided  hard  boiled  white  of  egg.  (The  white  of  egg  can  be  easily  pre- 
pared according  to  the  method  given  under  the  pepsin  test.)  Make  one 
of  the  tubes  slightly  acid  by  the  addition  of  dilute  hydrochloric  acid, 
enough  to  amount  to  0.2  or  0.3  per  cent.  Now  place  all  of  them  in  water 
kept  at  40°  C.  At  the  end  of  half  an  hour  remove  one  of  the  alkaline 
tubes,  and  note  that  it  still  contains  unaltered  coagulated  albumin.  Test 
the  liquid  for  albumoses  and  peptones  as  given  above.  After  another  half 
hour,  test  a  second  tube  (after  filtration).  It  will  be  observed  that  as 
the  coagulated  protein  disappears  peptones  become  more  abundant. 

Allow  one  of  the  alkaline  tubes  to  remain  several  hours  at  a  tempera- 
ture of  40°  C.     In  time  it  develops  a  disagreeable  odor,  due  to  the  pres- 


1/6  PHYSIOLOGICAL    CHEMISTRY. 

ence  of  indol  formed.  The  tube  containing  the  hydrochloric  acid  kept 
several  hours  at  40°  C.  does  not  show  the  effect  of  digestion,  indicating 
that  an  acid  medium  does  not  suffice  for  the  converting  activity  of  the 
pancreatic  ferment.  A  minute  trace  of  acid,  below  about  0.05  per  cent, 
does  not  appear  to  check  the  action. 

To  readily  recognize  the  final  products  of  the  pancreatic  digestion  of 
proteins  it  is  necessary  to  start  with  larger  quantities  of  materials  than 
are  given  above. 

An  experiment  made  as  above  shows  at  first  digestion  and 
finally  bacterial  putrefaction  as  disclosed  by  the  indol  odor. 
A  better  idea  of  some  of  the  products  formed  in  digestion  may 
be  secured  by  operating  as  follows : 

Ex.  Mince  50  gm.  of  fresh  fibrin  and  25  gni.  of  pancreas,  mix  and 
cover  the  mixture  with  250  cc.  of  alkaline  thymolized  water,  the  thymol 
being  added  to  check  too  rapid  putrefaction.  Keep  the  mixture  at  40° 
two  or  three  days  in  a  closed  vessel,  the  mass  being  frequently  shaken  or 
stirred.  At  the  end  of  the  digestion  the  alkali  of  the  mixture  is  neutral- 
ized with  a  faint  excess  of  acetic  acid,  after  which  it  is  boiled  in  a  porce- 
lain dish  and  filtered.  Some  of  the  fibrin  may  remain  and  there  will 
always  be  some  fat  to  separate  by  the  filtration.  The  filtrate  is  used  for 
the  identification  of  important  products,  some  of  which  are  readily  recog- 
nized, while  others  are  not. 

PRODUCTS  OF  DIGESTION. 

Tryptophane.  This  name  is  given  to  a  peculiar  product 
or  mixture  of  products  found  in  a  pancreatic  digestion  like  the 
above.  It  is  characterized  by  giving  a  marked  violet  red  color 
when  mixed  with  a  little  chlorine  water  or  bromine  water. 
The  composition  of  the  tryptophane  is  not  yet  known,  but  on 
treatment  with  alkalies  at  the  fusion  temperature  a  mixture 
of  several  complex  aromatic  products,  including  indol  and 
pyrrol,  is  obtained.  Quite  recently  the  name  tryptophane  has 
been  given  to  one  of  the  constituents  of  this  mixture  which 
has  the  formula  C11H12N0O2  and  which  has  been  shown  to  be 
skatol-a-aminoacetic  acid. 

In  a  concentrated  solution  the  addition  of  bromine  or  chlor- 
ine produces  a  precipitate.  This  may  be  redissolved  only  in 
a  very  considerable  excess  of  water.  The  solution  does  not 
yield  the  protein  reactions  at  all,  from  which  it  follows  that 


PRODUCTS    OF    PANCREATIC    DIGESTION.  1/7 

the  body  is  an  advanced  decomposition  product.  The  sub- 
stance is  sometimes  called  proteinochromogen. 

Ex.  To  recognize  the  chromogen  or  tryptophane  use  two  or  three  cc. 
of  the  above  filtrate  from  the  digestion  experiment.  Add  to  the  liquid 
some  bromine  water,  drop  b}-  drop,  shaking  after  each  addition.  Finally 
the  desired  color  appears. 

Tyrosine  and  Leucine.  These  important  amino  acids  have 
already  been  referred  to  when  the  decomposition  products  of 
proteins  were  described.  They  are  formed  abundantly  in  a 
prolonged  digestion  like  the  above  and  may  be  easily  recog- 
nized.    Tyrosine  is  paraoxyphenyl-a-aminopropionic  acid, 

/OH 
C  H    '^^ 

'  \CH2CH(NHOCOOH 

and  is  formed  from  most  of  the  protein  bodies  on  digestion. 
It  is  not  formed  in  appreciable  quantity  from  gelatin.  Leu- 
cine is  regarded  as  a  caproic  acid  derivative,  or  possibly 
as  a-aminoisobutylacetic  acid  (CH3)2CH.CH2.CH(NH2)- 
COOH.  It  is  one  of  the  most  common  of  the  protein  cleavage 
products,  and  is  formed  from  gelatin  also.  Both  of  these  sub- 
stances are  but  slightly  soluble  in  cold  water  and  may  be  eas- 
ily separated  in  crystalline  form. 

Ex.  To  recognize  the  two  amino  acids  in  the  digestion  mixture  pro- 
ceed as  follows :  Concentrate  the  bulk  of  the  liquid  to  a  volume  of  25  cc. 
and  allow  it  to  stand  in  a  cold  place  several  days.  At  the  end  of  this  time 
filter  through  fine  muslin  or  a  coarse  filter  paper.  The  granular  mass 
so  collected  contains  some  tyrosine  while  the  bulk  of  the  leucine  remains 
in  the  filtrate.  Examine  the  residue  first.  Wash  it  into  a  beaker  with 
a  little  cold  water,  allow  to  settle,  decant  and  wash  again  by  decantation. 
Then  add  a  larger  volume  of  water  and  enough  ammonia  to  give  a  marked 
odor.  Heat  to  boiling  and  filter  hot.  The  tyrosine  dissolves  in  the  alka- 
line liquid.  Concentrate  the  filtrate  until  the  odor  of  ammonia  has  dis- 
appeared and  allow  to  cool;  crystals  of  tyrosine  separate. 

Examine  some  of  these  under  the  microscope.  The  appearance  is  that 
of  bunches  or  sheaves  of  fine  needles.  These  needles  may  be  dissolved  in 
alkalies  and  also  in  hydrochloric  acid  on  the  slide,  which  behavior  distin- 
guishes them  from  other  somewhat  similar  crystalline  deposits. 

Millon's  Test.  A  very  distinctive  test  is  by  the  use  of  Millon's  reagent, 
which  has  been  already  illustrated.  Mix  a  little  of  the  crystalline  deposit 
with  some  water  and  Millon's  reagent  in  a  test-tube  and  apply  heat.     A 

13 


1/8  PHYSIOLOGICAL    CHEMISTRY. 

red  precipitate  forms  after  a  time  if  much  tyrosine  is  taken.  With  only 
a  minute  amount  a  red  color  only  may  result.  It  will  be  remembered  that 
this  reaction  is  not  confined  to  tyrosine  alone,  but  is  given  by  many  ben- 
zene derivatives  containing  a  hydroxyl  group  attached  to  the  nucleus. 
Hence  phenol  gives  the  test  distinctly. 

By  heating  a  little  of  the  crystalline  residue  with  2  or  3  cc.  of  strong 
sulphuric  acid  solution  follows.  On  adding  a  drop  of  formaldehyde  solu- 
tion a  red  color  is  produced  which  becomes  green  on  heating  further  with 
addition  of  some  glacial  acetic  acid. 

The  solution  left  after  filtering  off  the  tyrosine  is  concentrated  still 
more,  which  finally  causes  a  separation  of  leucine.  The  concentration  is 
continued  until  a  volume  of  about  5  cc.  is  reached.  Cr>'Stals  which  have 
separated  may  be  examined  under  the  microscope.  To  the  concentrated 
liquid  about  20  cc.  of  alcohol  is  added,  the  mixture  heated  on  the  water- 
bath  to  the  boiling  point  and  then  allowed  to  stand  until  cold.  It  is  then 
filtered.  The  filtrate  contains  most  of  the  leucine  present.  In  the  pre- 
cipitate there  is  peptone.  Evaporate  the  alcoholic  liquid  slowly  to  dry- 
ness, take  up  the  residue  with  water,  add  some  lead  hydroxide  (lead  oxide 
with  a  little  alkali),  boil  and  filter.  From  the  filtrate  remove  the  excess 
of  lead  by  means  of  hydrogen  sulphide;  filter  again  and  concentrate  the 
liquid  to  a  small  bulk  for  the  crystallization  of  the  leucine. 

Examine  some  of  the  leucine  crystals  under  the  microscope.  They  ap- 
pear as  spherical  bunches  of  very  fine  needles.  Often  the  needle  structure 
is  not  visible.  Hydrochloric  acid  and  weak  alkali  solutions  dissolve  the 
needles  on  the  slide. 

Leucine  gives  some  marked  chemical  tests.  Dissolve  some  of  the  crys- 
tals in  water,  add  sufficient  sodium  hydroxide  to  give  a  good  alkaline 
reaction  and  then  a  few  drops  of  copper  sulphate  solution.  The  precipi- 
tate of  copper  hydroxide  which  forms  at  first  redissolves,  giving  place  to 
a  blue  solution  containing  a  compound  of  leucine  and  copper. 

Leucine  may  be  oxidized  to  yield  valeric  acid.  On  this  behavior  a  test 
is  based.  To  some  of  the  crystalline  residue  containing  leucine  add  3 
drops  of  water  and  2  or  3  grams  of  solid  potassium  hydroxide.  Heat  in 
a  test-tube  until  the  alkali  melts.  The  leucine  decomposes,  giving  off  am- 
monia. Allow  the  mass  to  cool,  add  enough  water  to  dissolve  the  residue 
and  then  enough  dilute  sulphuric  acid  to  give  a  sharp  reaction.  On  apply- 
ing heat  the  odor  of  valeric  acid  becomes  evident.  Through  the  alkaline 
oxidation  carbon  dioxide  is  split  off. 

Albumoses  and  Peptones.  These  bodies  are  contained  in 
the  residue  insoluble  in  alcohol  left  above  after  separation  of 
the  leucine.  This  residue  is  washed  more  completely  with 
strong  alcohol  and  dissolved  finally  in  water.  The  solution 
may  be  tested  for  albumose  and  peptone  by  methods  already 
sriven. 


PRODUCTS    OF    PANCREATIC    DIGESTION.  1/9 

Indol  and  Skatol.  In  a  prolonged  pancreatic  digestion, 
especially  in  the  absence  of  the  protecting  thymol  or  chloro- 
form, these  bodies  are  always  formed.  Their  appearance  has 
nothing  to  do,  however,  with  the  enzymic  fermentation  which 
gives  rise  to  the  other  products.  They  are  always  products  of 
bacterial  decomposition  and  seem  to  be  produced  by  the  bac- 
teria from  some  of  the  enzymic  products.  Indol  has  the 
composition, 


/CH 


Skatol  is  the  methyl  derivative, 

^CHa 
CeH.<f       \CH. 

Pure  indol  is  a  crystalline  substance  melting  at  52°.     Skatol 

melts  at  95°.     Indol  is  oxidized  in  the  body  to  indoxyl,  which 

appears  in  part  in  the  urine  as  indican  or  potassium  indoxyl 

sulphate, 

CsHbN\ 

>S0.. 

Skatol  suffers  a  similar  change.  More  will  be  said  about  these 
reactions  later.  Although  these  bodies  are  not  true  pancreatic 
products,  it  may  be  well  to  illustrate  their  production  in  this 
place,  since  they  frequently  appear  in  pancreatic  digestions. 
An  experiment  will  shoAv  this. 

Ex.  Chop  fine  500  grams  of  meat  and  25  grams  of  pancreas  and  allow 
the  mixture  to  stand  exposed  a  day.  Then  mix  with  2  liters  of  water 
and  50  cc.  of  a  saturated  solution  of  sodium  carbonate,  place  in  a  flask 
and  keep  at  a  temperature  of  40°  through  about  10  days.  Then  transfer 
the  whole  mass  to  a  large  tin  or  copper  can  and  distil  off  most  of  the 
liquid.  For  a  complete  separation  500  cc.  of  water  should  be  added  at 
this  stage  and  this  distilled  also.  The  whole  of  the  distillate  is  now  acidi- 
fied with  hydrochloric  acid  and  divided  into  portions  of  300  cc.  each,  which 
are  shaken  out  thoroughly  in  a  separatory  funnel  with  ether.  For  the  first 
300  cc.  of  acid  liquid  about  200  cc.  of  ether  should  be  used.  The  extracted 
aqueous  layer  is  drawn  off  and  a  new  portion  of  300  cc.  added  to  the  same 


I  So  PHYSIOLOGICAL    CHEMISTRY. 

ether.  About  50  cc.  of  fresh  ether  must  also  be  added.  The  mixture  is 
thoroughly  shaken,  separated  as  before,  and  the  operation  repeated  until 
all  the  acidified  distillate  is  extracted.  The  ether  is  mixed  with  an  equal 
volume  of  water  and  enough  sodium  hydroxide  to  give  a  strong  reaction. 
The  alkali  combines  with  and  holds  the  volatile  acids  which  are  present 
while  indol  and  skatol  remain  in  the  ether  layer.  Separate  as  before, 
transfer  the  ether  to  a  flask  and  distil  at  a  low  temperature.  Drive  off 
three-fourths  of  the  ether  and  allow  the  remainder  to  evaporate  spontane- 
ously. It  will  not  be  necessary  to  purify  the  residue  in  any  way.  Dilute 
it  largely  with  water  and  apply  the  following  tests : 

Transfer  10  cc.  of  the  dilute  indol  solution  to  a  test-tube  and  add  i  cc. 
of  a  dilute  sodium  nitrite  solution,  mix  thoroughly  by  shaking  and  then 
pour  carefully  a  few  cc.  of  strong  sulphuric  acid  down  the  side  of  the 
tube  so  as  to  form  a  layer  below  the  other  liquid.  At  the  junction  of  the 
two  liquids  a  purple  red  color  is  formed,  which  changes  to  bluish  green 
on  neutralization  with  alkali.  This  test  is  similar  to  the  one  commonly 
employed  in  water  analysis  to  detect  the  presence  of  indol-producing  bac- 
teria. The  nitrite  solution  used  must  be  ver\-  weak,  preferablj-  not  over 
0.02  per  cent  in  strength. 

Another  test  is  performed  in  this  way.  A  splinter  of  soft  pine  wood 
is  moistened  with  strong  hydrochloric  acid  and  then  dipped  in  a  weak 
aqueous  solution  Of  indol.  The  wood  gradually  becomes  red.  With  much 
indol  the  color  becomes  deep  and  characteristic. 

A  characteristic  test  of  value  depends  on  the  formation  of  a  salt  of 
nitroso-indol.  Acidify  the  indol  solution  to  be  tested  with  nitric  acid  and 
then  add  a  few  drops  of  a  2  per  cent  solution  of  sodium  nitrite.  The 
nitrate  of  nitroso-indol,  CioHi3(NO)N2HN03,  forms  and  produces  a  red 
precipitate  if  much  indol  is  present.  If  the  indol  solution  is  weak  a  red 
color  only  forms.  By  adding  some  chloroform  and  shaking,  the  indol  may 
be  concentrated  in  the  junction  laj-er  between  the  two  liquids. 

By  adding  a  weak  solution  of  sodium  nitroprusside  to  an  indol  solution 
a  yellow  color  is  first  obtained.  The  addition  of  weak  sodium  hydroxide 
changes  this  to  violet,  which,  in  turn,  becomes  blue  by  acidifying  with 
acetic  acid.     This  is  known  as  Legal's  test. 

Skatol  fails  to  give  the  above  tests. 

OTHER  PRODUCTS   OF   TRYPTIC   DIGESTION. 

In  the  last  few  pages  a  brief  summary  of  some  of  the  most 
easily  recognized  of  the  products  formed  in  the  pancreatic 
digestion  of  proteins  has  been  given.  But  the  summary  is  by 
no  means  complete,  as  the  number  of  products  obtained  in  the 
protein  disintegration  is  far  larger  than  here  suggested.  Ac- 
cording to  the  older  views  of  peptic  and  pancreatic  digestion 
the  amphopeptone  of  the  stomach  passes  largely  into  antipep- 


PRODUCTS    OF    PANCREATIC    DIGESTION.  l8l 

tone  under  the  influence  of  trypsin.  But  the  amphopeptone 
contains  some  of  the  heini  group  also  and  this  under  energetic 
pancreatic  action  finally  yields  cr5^stalline  products  no  longer 
capable  of  giving  the  biuret  reaction.  The  anti  group  as 
finally  found  in  the  antipeptone  was  long  supposed  to  be  a 
permanent  end  product,  not  subject  to  further  digestive 
change.  But  this  view  seems  to  be  erroneous.  It  has  been 
already  stated  that  pancreatic  digestion  as  distinguished  from 
peptic  digestion  may  be  carried  so  far  that  nothing  is  left 
which  gives  the  biuret  reaction.  When  the  peculiar  group  on 
which  this  reaction  depends  drops  out,  the  whole  molecular 
structure  is  so  badly  shattered  that  nothing  which  may  be  con- 
sidered as  closely  related  to  the  proteins  remains. 

The  question  then  comes  up,  what  are  the  final  products  of 
tryptic  action?  Numerous  investigations  of  comparatively 
recent  date  give  us  more  or  less  satisfactory  answers  to  this 
question. 

Carnic  Acid.  From  a  number  of  sources,  particularly  from 
beef  extract,  Siegfried  has  obtained  a  product  which  he  calls 
carnic  acid  and  which  seems  to  have  a  constant  composition 
represented  by  the  formula,  C10H15N3O5.  In  the  muscles  and 
in  milk  this  acid  is  held  to  exist  in  combination  with  phos- 
phorus in  the  so-called  phosphocarnic  acid,  the  iron  combina- 
tion of  which  is  known  as  carniferrin.  The  true  carnic  acid 
gives  the  biuret  reaction,  but  the  Millon  reaction  only  faintly. 
It  forms  crystalline  salts  with  copper,  barium,  zinc  and  other 
metals.  Its  solutions  are  precipitated  by  phospho-tungstic 
acid,  tannic  acid  and  picric  acid,  but  not  by  ammonium 
sulphate. 

Siegfried  considers  this  carnic  acid  as  identical  with  anti- 
peptone,  and  a  large  number  of  papers  have  been  published  in 
support  of  the  theory.  The  carnic  acid  must  be  made  up  of 
groups  still  holding  the  biuret  complex,  in  which  leucine,  tyro- 
sine, the  hexone  bases  and  perhaps  other  bodies  are  all  joined 
in  some  way  not  yet  understood. 

The  Hexone  Bases  and  Other  Bodies.     The  Siegfried 


1 82  PHYSIOLOGICAL    CHEMISTRY. 

view  has  not  been  generally  accepted  as  a  whole.  Other  in- 
vestigators appear  to  have  shown  that  what  Siegfried  took  for 
a  single  body  of  constant  composition  is  really  a  mixture  of 
substances,  yielding  pretty  constant  results  on  analysis.  In 
this  mixture  the  hexone  bases,  arginine,  lysine  and  histidine, 
are  important  components.  These  are  all  diamino  acids  with 
six  carbon  atoms  and,  because  of  their  constant  occurrence  in 
digestive  mixtures  and  other  products  of  protein  decomposi- 
tion, they  must  be  looked  upon  as  essential  factors  in  the  pro- 
tein structure.  Leucine  and  tyrosine  always  seem  to  accom- 
pany the  hexones  in  these  decompositions. 

Although  by  prolonged  digestion  products  are  reached 
which  do  not  give  the  biuret  reaction,  it  is  claimed  by  Fischer 
and  others  in  recent  work  that  residues  remain  which  are  still 
relatively  complex.  The  name  polypeptides  has  been  given 
by  Fischer  to  such  residues,  and  their  relations  to  chemical 
substances  of  definite  composition  pointed  out.  But  even 
these  may  be  finally  broken  down  into  amino  acids. 

Synthesis  of  Polypeptides.  In  the  last  few  years  a  number  of  these 
polypeptides  have  been  produced  by  several  synthetic  processes.  Among 
such  bodies  described  by  Fischer  the  following  may  be  cited  as  illustra- 
tions : 

DiGLYCYLGLYCiNE,  NH2CH2CO  "  NHCH2CO  "  NHCH=COOH.  This  is  a 
tripcptide  and,  as  the  formula  shows,  is  formed  by  a  condensation  of  three 
groups  of  aminoacetic  acid. 

Al.\nvlglvcylglycine,  CH3CHNH2CO  ■  NHCH:CO  •  NHCH=COOH.  In 
this  compound  alanine,  a-aminopropionic  acid,  is  one  of  the  groups  brought 
into  the  combination  with  glycine. 

Pheny'l.\lanylglycylglycine,  C6HBCH2CHNH2CO  •  NHCHjCO  ■  NH 
CH2COOH.  This  body  is  of  interest  because  of  the  occurrence  of  phenyl- 
alanine among  the  commoner  protein  cleavage  products,  where  reagents 
are  used.  Residues  containing  this  group  appear  to  be  much  more  re- 
sistant toward  tryptic  fermentation. 

Leucy'Lproline.     Proline  =  a-pyrrolidine  carboxylic  acid. 

CHav  /CH2— CH. 

>CH-CH2CH-CO-N< 
CH3/  I  ^CH  —  CH2 

NH2  [ 

COOH 

In  this  case  the  synthesis  of  leucine  and  the  pyrrohdine  carboxylic  acid 
has  been  made.     In  trj^jsin  digestion  residues  containing  the  latter  body 


PRODUCTS    OF    PANCREATIC    DIGESTION.  I  S3 

along  with  phenylalanine  seem  to  be  characteristic,  especially  where  casein 
is  used.  But  these  residues  are  easily  decomposed  by  hydrochloric  acid 
with  separation  of  the  constituent  amino  acids.  It  is  likely  that  much 
more  complex  bodies  will  be  secured  by  further  extension  of  the  processes. 

The  hexone  bodies  as  end  products  of  definite  composition 
are  of  great  theoretical  importance  because  of  their  relation 
to  the  protamines  referred  to  in  a  former  chapter.  Some  of 
these  protamines  break  down  almost  quantitatively  into  argi- 
nine  and  the  other  hexones,  so  that  the  latter  may  well  be 
looked  upon  as  nucleus  structures  which  unite,  with  loss  of 
water,  to  form  the  more  complex  molecules.  These  diamino 
acids  seem  to  bear  about  the  same  relation  to  the  peptones  and 
proteins  that  sugar  bears  to  dextrin  and  starch.  As  in  the 
hydrolysis  of  starch  the  nature  of  the  end  product  depends  on 
the  nature  of  the  agent  of  cleavage,  so  in  proteolysis  the  same 
thing  is  true ;  acids  and  enzymes  work  nearly  in  the  same  way, 
but  not  absolutely. 

In  this  connection  it  should  be  pointed  out  that  Siegfred  has 
separated  by  a  somewhat  peculiar  method  of  treatment  a  num- 
ber of  bodies  which  he  calls  trypsin-fibrin  peptones  and  pepsin- 
fibrin  peptones  which  may  be  represented  by  the  following 
formulas : 

trypsin  antipeptone  a  C10H17N3O5 
trypsin  antipeptone  ^  C11H10N3O5 
pepsin  peptone  a  CaiHaiNeOg 

pepsin  peptone  ^  C21H36N6O10 

The  pepsin  peptone  a  seems  to  be  related  to  the  antipeptones 
in  this  way : 

C^iHs^NeOs,  -I-  H2O  =  CioHitNsO,  +  CUH19N3O5 

It  is  urged  by  Siegfried  that  the  constant  optical  rotation  of 
these  various  products  is  a  satisfactory  evidence  of  their  con- 
stant composition.  In  connection  wath  these  formulas  the 
formulas  of  the  hexone  bodies  may  be  recalled : 

histidine,  CeHsNaOs 
arginine,  CsHuNiOa 
lysine,        CeHuNsO^ 


1 84  PHYSIOLOGICAL    CHEMISTRY. 

These  compounds  are  relatively  much  simpler  than  the  Sieg- 
fried peptones  and  might  readily  be  derived  from  them,  with 
separation,  at  the  same  time,  of  still  smaller  molecules. 

The  conception  of  "  end  product  "  in  tryptic  digestion  is 
evidently  a  somewhat  indefinite  one.  Certainly  in  the  animal 
body  the  digestive  cleavage  cannot  extend  to  the  production 
of  these  small  molecules  which  would  doubtless  be  useless  for 
nutrition.  What  is  obtained  in  artificial  digestions  depends 
largely  on  the  time  given  and  the  activity  of  the  enzyme  em- 
ployed; the  term  "end  product"  is  therefore  wholly  relative. 

TOXICITY   OF  DIGESTIVE   PRODUCTS. 

It  has  been  known  for  a  long  time  that  some  of  the  artificial 
digestive  products  when  injected  into  the  circulation  directly 
exhibit  a  marked  toxic  action.  Experiments  in  this  direction 
have  been  made  mostly  on  dogs,  and  the  material  employed 
was  usually  the  commercial  product  known  as  Witte's  peptone, 
which  contains  some  real  peptone  with  a  much  larger  portion 
of  proteoses.  In  experimenting  on  dogs  it  has  been  found  that 
500  milligrams  for  each  kilogram  of  body  weight  was  sufficient 
to  produce  a  marked  decrease  in  blood  pressure.  The  clotting 
power  of  the  blood  is  at  the  same  time  greatly  impaired  or 
even  destroyed.     Larger  injections  may  produce  death. 

This  effect  varies  with  the  nature  of  the  peptone  used.  In 
the  earlier  investigations  it  was  assumed  that  the  commercial 
peptone  was  a  fairly  definite  product,  and  in  addition,  a  real 
peptone.  It  is  now  well  known  that  this  was  not  the  case,  and 
in  repeating  the  experiments  with  products  made  by  modern 
methods  it  has  been  recognized  that  the  purified  pancreas  pep- 
tone, which  is  a  much  more  highly  converted  product  than  the 
Witte  peptone,  is  practically  inert.  The  toxicity  is  apparently 
due  to  the  presence  of  one  or  more  of  the  lower  albumoses. 
A  large  number  of  experiments  carried  out  by  Chittenden  and 
others  show  the  effects  obtainable  with  the  different  classes  of 
digestion  products.  The  two  principal  effects,  lowering  of 
arterial  pressure  and  delaying  or  impeding  coagulation,  are 


PRODUCTS    OF    PANCREATIC    DIGESTION.  1 85 

independent  of  each  other.  The  first  appears  to  be  due  to  local 
action  on  the  endings  of  the  vasomotor  nerves  in  the  blood 
vessels  of  such  a  character  that  unusual  vascular  dilatation  fol- 
lows. The  effect  on  coagulation  appears  to  be  due  to  a  pecu- 
liar action  of  the  albumoses  on  the  white  blood  corpuscles  in 
which  the  latter  are  rapidly  disintegrated.  The  disintegra- 
tion products  are  of  two  kinds,  the  one  hastening  and  the  other 
retarding  coagulation.  It  appears  that  the  liver  cells  combine 
with  the  products  which  hasten  coagulation,  so  that  the  final 
effect  is  to  leave  the  blood  in  a  condition  where  coagulation  is 
very  slow.  No  satisfactory  chemical  explanation  can  be 
offered  for  these  phenomena. 

It  has  been  suggested  recently  that  the  fully  purified  albu- 
moses, like  the  two  pancreas  peptones,  are  non-toxic.  Investi- 
gations by  Pick  seem  to  show  that  the  producing  enzyme, 
which  is  hard  to  separate  from  the  resultant  albumose  or  pep- 
tone, is  probably  responsible  for  the  observed  phenomena. 

THE  CARBOHYDRATE  DIGESTION. 

The  pancreas  furnishes  an  enzyme  called  amylopsin  or  pan- 
creatic diastase  which  acts  on  starch  or  dextrin  to  form  sugar. 
Beginning  with  starch  we  have  the  gradual  formation  of  mal- 
tose by  hydrolysis.  It  has  been  already  pointed  out  that  this 
is  not  a  simple  process  but  one  which  takes  place  in  several 
stages,  various  kinds  of  "  dextrins  "  coming  in  between  the 
original  starch  and  the  final  sugar.  In  addition  to  the  enzyme 
which  forms  the  malt  sugar  the  pancreas  furnishes  a  "  mal- 
tase  "  which  converts  this  malt  sugar  into  glucose.  The  action 
may  be  very  well  shown  by  means  of  the  glycerol  extracts  of 
pancreas  described  some  pages  back  under  the  head  of  tryptic 
digestion. 

Ex.  Prepare  a  starch  paste  with  5  gm.  of  starch  to  100  cc.  of  water. 
Mix  10  cc.  of  this  paste,  after  cooHng,  with  5  cc.  of  the  pancreatic  extract, 
warm  to  a  temperature  of  35°-40°  C.  and  notice  that  the  paste  soon 
becomes  thin  and  nearly  clear.  After  a  time  test  for  sugar.  Repeat  the 
experiment,  using  pancreatic  extract  which  has  been  boiled  before  mixing 
with  the  starch.     The  sugar  reaction  now  fails  to  appear,  showing  that 


1 86  PHYSIOLOGICAL    CHEMISTRY. 

high  temperature  destroys  the  activity  of  the  enzyme,  as  in  the  case  of 
sahva.  Note  in  the  solution  of  the  starch  the  dextrin  stages  which  may 
be  followed  by  the  iodine  test.  For  the  complete  conversion  of  the 
amount  of  starch  here  taken  some  hours  may  be  required.  This  depends 
on  the  strength  of  the  pancreas  extract. 

It  is  well  to  vary  the  experiment  by  employing  some  fresh  minced  gland 
in  place,  of  the  extract.     The  pancreas  powder  may  also  be  used. 

We  have  then  the  two  principal  reactions  here,  the  forma- 
tion of  mah  sugar  and  the  inversion  of  the  same.  It  is  not 
possible  to  isolate  the  enzyme  which  produces  the  one  reaction 
from  that  which  gives  rise  to  the  other,  but  that  both  are  prod- 
ucts of  the  cells  of  the  pancreas  has  been  satisfactorily  shown 
against  the  view  that  the  inxerting  enzyme  is  furnished  by  the 
so-called  intestinal  juice.  It  may  be  recalled  that  both  reac- 
tions are  hydrolytic. 

It  is  likely  that  the  living  gland  does  not  contain  the  active 
ferment  itself  but  a  proferment  or  zymogen,  which  becomes 
active  after  the  secretion  has  passed  into  the  intestine.  In 
the  minced  gland  the  change  appears  to  take  place  through 
the  agency  of  air  and  moisture.  There  is  a  marked  differ- 
ence in  the  activity  of  the  glands  of  different  animals,  which 
fact  is  practically  recognized  by  the  manufacturers  of  the  com- 
mercial products.  The  pancreas  of  the  hog  furnishes  an  en- 
zymic  mixture  richer  in  the  starch  digesting  agents,  while  the 
beef  pancreas  seems  to  be  most  active  in  the  digestion  of 
proteins. 

As  the  proteins  are  prepared  practically  for  final  absorption 
from  the  intestine  by  the  action  of  trypsin,  so  the  remains  of 
the  carbohydrates  are  brought  into  the  proper  final  condition 
by  the  amylopsin  and  maltase;  at  any  rate  the  starches  are 
so  prepared,  and  maltose  from  any  source  also.  But  as  to 
cane  sugar  and  milk  sugar  there  appears  to  be  some  little 
doubt,  several  authors  claiming  that  the  pancreas  does  not  con- 
tain lactase  or  invertase,  but  that  the  changes  in  these  sub- 
stances, w^hen  not  already  accomplished  by  the  acid  gastric 
juice,  take  place  through  the  agency  of  the  enzymes  of  the 
intestine. 


PRODUCTS    OF    PANCREATIC    DIGESTION,  I  8/ 

THE  ACTION  OF  THE  PANCREAS  ON  FATS. 

In  the  general  discussion  of  the  subject  of  enzymes  it  was 
shown  that  a  certain  product  of  the  pancreas  called  steapsin 
or  lipase  is  active  in  splitting  neutral  fats  into  glycerol  and 
acid.  This  is  a  true  change  by  hydrolysis  and  in  effect  is 
similar  to  the  splitting  by  water  alone  at  an  elevated  tempera- 
ture. In  the  pancreas  the  reaction  may  not  be  complete,  but 
may  extend  only  to  the  separation  of  one-third  of  the  acid  as 
illustrated  by  this  equation  for  stearin : 

r  C18H35O2  r  OH 

aaJ  CisH350.  +  HOH  =  C3H5^  C1SH35O2  +  HCXSH3.O2 

I C1SH35O2  L  C13H35O2 

This  amount  of  liberated  acid  combining  with  the  sodium 
carbonate  of  the  intestinal  juices  produces  a  soap  which  in 
turn  aids  in  the  emulsification  of  the  rest  of  the  fat  and  thus 
prepares  for  its  passage  through  the  intestinal  walls.  On  the 
other  hand,  certain  writers  maintain  that  the  fat  must  be  essen- 
tially all  split  before  absorption  is  possible.  The  fatty  acid 
and  glycerol  pass  through  the  intestinal  walls  directly  and 
recombine.  The  change  in  this  case  would  follow  through 
the  typical  equation : 

C3H5(Cl8H3502)3+3H20=C3H5(OH)3  +  3Cl8H3602 

The  behavior  of  the  pancreas  may  be  shown  by  experiment, 
but  for  this  purpose  it  is  much  better  to  use  the  fresh  pancreas 
than  to  depend  on  extracts.  The  lipase  seems  to  be  soluble 
in  glycerol  to  some  extent,  but  unless  the  fresh  gland  is  em- 
ployed for  the  extraction  the  result  may  be  unsatisfactory. 
These  points  may  be  tested  by  the  student : 

Ex.  Rub  up  a  part  of  a  pancreas  with  some  fine,  clean  sand  in  a  mor- 
tar to  bring  it  into  the  condition  of  a  creamy  paste.  Add  some  water, 
mix  thoroughly,  and  use  this  for  the  tests.  Next  melt  some  butter  to 
allow  the  curd  and  salt  to  settle.  Collect  the  clear  butter  fat.  Mix  a 
few  grams  of  the  fat  with  an  equal  volume  of  the  pancreas  paste,  add 
some  water  and  a  few  drops  of  chloroform  as  preservative.  Keep  the 
mixture  at  a  temperature  of  40°  through  a  period  of  several  hours  or  over 
night,  and  observe  that  it  gradually  becomes  acid  through  the  liberation 


I  88  PHYSIOLOGICAL    CHEMISTRY. 

of  butyric  acid  from  the  butyrin.  This  may  be  shown  qualitatively  by 
means  of  the  reaction  with  rosolic  acid,  a  slightly  alkaline  solution  with 
red  color  changing  to  yellow  on  addition  of  some  of  the  pancreas-fat 
mixture.  It  may  also  be  shown  by  adding  a  few  drops  of  phenol-phthalein 
to  some  of  the  mixture  and  then  gradually  very  weak  sodium  hydroxide 
until  the  alkaline  reaction  is  secured.  With  a  standard  alkali  solution 
the  volume  used  becomes  a  measure  of  the  amount  of  acid  set  free. 

In  this  form  of  the  experiment  the  butter  fat  is  more  readily  decomposed 
than  are  the  more  solid  neutral  fats.  Indeed  the  lighter  esters  are  fre- 
quently used  to  detect  vegetable  lipase  through  the  same  general  reaction. 
If  in  the  experiment  the  butter  fat  used  is  not  neutral  to  begin  with,  it  is 
best  to  add  a  few  drops  of  rosolic  acid  and  then  very  weak  alkali  until 
the  color  just  changes  to  red.  Lipase  is  destroyed  by  heat  as  are  the 
other  enzymes. 

Ex.  The  emulsifying  power  of  the  pancreas  may  be  shown  also.  Grind 
some  fresh  pancreas  to  a  thin  paste  with  a  little  water.  Add  several 
grams  of  this  mixture  to  some  perfectly  neutral  refined  cotton-seefl  oil, 
about  10  cc,  in  a  warm  mortar  and  rub  thoroughly  with  a  pestle.  After 
a  considerable  time  an  emulsion  forms  which  will  bear  dilution  with  much 
water.  With  common  oil  containing  a  little  free  fatty  acid  the  emulsion 
forms  more  rapidly,  but  in  this  case  the  reaction  may  be  largely  due  to 
the  formation  of  soap  first,  from  the  combination  of  the  fatty  acid  and 
alkali  of  the  pancreatic  secretion.  The  experiment  would  therefore  fail 
to  show  the  presence  of  an  enzyme  as  fat  splitter.  For  success  here  a 
fresh  pancreas  is  necessary. 

A  pure  neutral  fat  suitable  for  such  experiments  may  be  obtained  by 
adding  a  little  caustic  soda  solution  to  some  refined  cotton-seed  oil  and 
then  ether.  On  shaking  thoroughly  the  neutral  fat  dissolves  in  the  ether, 
leaving  the  soap  formed  and  excess  of  alkali  undissolved,  practically.  The 
fat-ether  layer  is  poured  off,  shaken  several  times  with  water  for  the 
removal  of  traces  of  soap  or  alkali,  and  then  slowly  evaporated.  Neutral 
fat  is  left  after  the  volatilization  of  the  ether. 

In  these  emulsification  reactions  the  pancreatic  secretion  is 
assisted  by  the  alkahne  bile.  According  to  the  theory  of  the 
formation  of  soaps,  as  a  prehminary  to  absorption  from  the  in- 
testines, the  bile  must  act  as  a  very  important  factor,  as  its 
alkali  would  be  needed  for  the  purpose.  In  addition  the  bile 
has  a  distinct  solvent  action  on  fatty  acids  which  may  be  of 
help  in  the  ultimate  passage  of  the  fat  products  from  the 
intestine.  The  general  nature  of  the  bile  products  will  be 
discussed  later. 


PRODUCTS    OF    PANCREATIC    DIGESTIOX.  1 89 

THE  FUNCTION  OF  THE  INTESTINAL  JUICE. 

Closely  related  to  the  action  of  the  pancreatic  diastases  is 
the  behavior  of  certain  enzymes  entering  the  small  intestine 
from  other  sources,  especially  from  the  glands  of  Lieberkiihn. 
As  these  enzymes  seem  to  follow  up  and  complete  the  pan- 
creatic digestion,  they  may  be  briefly  mentioned  here.  It 
should  be  said  first,  however,  that  any  specific  digestive  action 
due  to  ferments  in  the  secretion  of  these  glands  was  for  a  long 
time  denied,  but  there  appears  now  to  be  no  further  question 
as  to  the  actual  behavior  of  the  secretion  in  this  respect. 

Character  of  the  Secretion.  The  flow  into  the  intestine 
from  the  Lieberkiihn  glands  consists  of  a  thin  serum-like 
liquid,  holding  in  solution  protein  bodies  and  salts.  The  re- 
action is  strongly  alkaline  because  of  the  presence  of  sodium 
carbonate.  The  amount  of  this  is  sufficient  to  give  rise  to  an 
evolution  of  carbon  dioxide  when  an  acid  is  added  to  the  secre- 
tion collected  by  a  fistula.  This  alkali  is  doubtless  important 
in  two  ways ;  it  aids  in  the  emulsification  of  fats,  and  also  helps 
in  the  neutralization  of  the  remaining  hydrochloric  acid  from 
the  gastric  juice  carried  into  the  intestine  with  the  chyme 
current. 

The  ferments  appear  to  have  little  or  no  action  on  fats  or 
proteins,  but  work  on  the  residues  of  carbohydrates  only. 
Some  chemists  claim  to  find  in  the  intestinal  juice  a  slight 
starch-digesting  power;  others  deny  that  such  a  behavior  is 
possible  and  limit  the  activity  of  the  secretion  to  the  inversion 
of  certain  sugars,  especially  cane  sugar  and  malt  sugar.  In- 
deed some  authors  go  so  far  as  to  urge  that  all  of  the  inversion 
processes  taking  place  in  the  intestine  are  brought  about  in  this 
way,  while  the  pancreas  can  produce  malt  sugar  only.  Inves- 
tigations of  this  kind  are  attended  with  considerable  difliculty, 
which  fact  must  be  kept  in  mind  when  attempting  to  draw 
conclusions  from  apparently  contradictory  statements,  such  as 
are  quoted  above.  All  recent  investigations  have  shown  this, 
that  while  the  intestinal  juice  may  not  be  the  sole  agent  of  in- 
version, it  is  certainly  an  important  agent  in  this  direction. 


190  PHYSIOLOGICAL    CHEMISTRY. 

The  ferments  present  are  evidently  of  two  types:  one  resem- 
bling the  invertase  of  cane  sug^r  already  described,  while  the 
other  is  of  the  maltase  type. 

The  Secretion  from  Brunner's  Glands.  The  collection  of 
the  product  from  these  small  glands  offers  considerable  tech- 
nical difficulty,  and  until  recently  no  very  clear  statements 
were  found  in  the  literature  as  to  the  exact  nature  of  the  secre- 
tion. By  taking  special  precautions,  however.  Glaessner  suc- 
ceeded in  securing  the  secretion  free  from  other  fluids,  and  has 
found  that  it  possesses  marked  proteohiiic  properties  in  solu- 
tions of  all  reactions.  The  digestion  of  protein  is  carried  to 
the  stage  where  tr^-ptophane  may  be  easily  recognized.  The 
name  pseudopepsin  may  be  given  to  the  active  enzyme. 

Erepsin.  Comparatively  recently  a  ferment  called  erepsin 
has  been  described  by  Cohnheim  in  the  intestinal  juice.  It 
does  not  digest  the  true  proteins  but  has  the  power  of  splitting 
albumoses  and  peptones  as  far  as  the  mono  and  diamino  acids. 
This  enzyme  may  have  importance  as  an  intercellular  ferment 
and  be  concerned  in  preparing  the  protein  digestive  products 
for  subsequent  synthesis. 

Enterokinase.  This  name  has  been  given  to  a  ferment- 
like body  which  occurs  in  the  intestinal  juice  and  which  has 
the  power  of  activating  tr\-psinogen.  Without  the  presence 
of  this  activator  it  is  held  by  some  recent  writers  that  trypsin 
is  not  formed  and  therefore  cannot  digest  protein.  The  en- 
terokinase is  not  a  digestive  agent  itself. 

In  the  brief  discussions  of  the  last  few  chapters  it  has  been 
shown  in  a  general  way  how  the  important  classes  of  food 
stuffs  through  the  action  of  enzymes  in  different  parts  of  the 
body  are  gradually  brought  into  a  condition  suitable  for  as- 
similation and  absorption.  They  have  undergone  digestion 
and  are  ready  to  be  carried  through  the  intestinal  walls  into 
the  blood  stream  to  be  used  as  food  for  the  building  up  of 
the  body  or  as  oxidation  material  for  the  production  of  me- 
chanical energ}-  and  heat.  These  various  digestive  processes 
differ  in  many  ways,  but  they  have  this  important  element  in 


PRODUCTS    OF    PANCREATIC    DIGESTION.  IQI 

common  which  must  be  kept  in  mind :  they  are  essentially 
hydrolytic  in  character,  the  addition  of  water  by  the  enzymes 
being  the  essential  feature  in  all  of  them.  We  have  next  to 
consider  some  changes  in  the  intestines  in  which  hydrolysis 
does  not  play  an  important  part. 


CHAPTER    X. 

CHANGES  IN  THE  INTESTINES.     THE  FECES. 

In  an  ideally  normal  condition  of  the  alimentary  canal  after 
the  completion  of  the  digestive  processes  described  in  the  last 
chapters,  there  should  be  practically  nothing  left  finally  in  the 
intestines  but  residues  of  non-nutritive  value,  along  with 
broken  down  products  from  the  digestive  agents  themselves. 
Every  trace  of  sugar  or  starch  should  have  been  brought  into 
the  form  of  a  monosaccharide  and  absorbed ;  every  particle  of 
fat  should  have  been  hydrolyzed  or  emulsified  and  then  car- 
ried into  the  lacteal  circulation ;  while  the  proteins  should  have 
reached  the  form  of  higher  albumoses  or  peptones  and  have 
been  likewise  absorbed.  The  actual  situation  approaches  this 
ideal  condition  only  approximately.  In  the  first  place  the 
foods  we  consume  are  not  absolutely  pure  fats,  carbohydrates 
or  proteins.  They  all  contain  some  mineral  matter  which  may 
escape  the  various  digestive  actions,  and  they  usually  contain 
certain  organic  substances  which  are  only  partially  digestible. 
Some  vegetable  foods,  for  example,  contain  relatively  large 
quantities  of  cellulose,  which  is  a  body  related  to  the  carbo- 
hydrates but  which  is  not  attacked  by  the  weak  digesting  en- 
zymes. In  the  foods  of  animal  origin  there  are  likewise  sub- 
stances which  are  very  difficult  of  digestive  hydrolysis.  This 
is  true  of  some  of  the  albuminoids;  horn-like  substances,  for 
example,  are  practically  not  attacked,  while  the  cartilaginous 
and  similar  bodies  are  but  slowly  changed.  From  foods  con- 
taining portions  of  such  compounds  a  residue  would  always 
be  left  therefore,  and  in  the  case  of  poor,  cheap  meat  this  resi- 
due might  be  considerable. 

OTHER  FERMENTATIONS. 

Bacterial  Processes.  But  the  case  is  complicated  by  other 
considerations.     Our  foods  carry  hosts  of  acid  and  putrefac- 

192 


CHANGES    IN    INTESTINES.       THE    FECES.  1 93 

tive  ferments  with  them;  and  some  of  these  at  times  work 
through  the  stomach  into  the  intestine,  where  they  start  reac- 
tions of  their  own.  Just  what  changes  take  place  in  the  small 
intestine  depends  on  the  character  of  the  food.  Following  the 
alkaline  zone  where  the  pancreatic  secretion,  the  bile  and  intes- 
tinal juice  rapidly  effect  the  changes  already  described,  there 
is  a  zone  of  acid  or  neutral  reaction  where  certain  fermentation 
processes  of  bacterial  origin  take  place.  If  the  food  is  rich 
in  carbohydrates  this  fermentation  may  be  considerable,  result- 
ing in  the  formation  of  appreciable  quantities  of  lactic,  butyric 
and  acetic  acids.  The  liberation  of  these  acids  at  this  stage 
is  a  matter  of  very  considerable  importance,  since  it  prevents 
the  breaking  up  of  not  yet  absorbed  protein  by  bacterial  putre- 
faction. If  the  acids  were  not  present  bacteria  would  reach 
the  small  intestine  in  enormous  numbers  from  the  large  intes- 
tine and  greatly  modify  the  conditions  there.  While,  along 
with  the  acid-forming  bacteria,  a  few  others  are  always  pres- 
ent in  the  small  intestine,  the  real  putrefactive  changes  do  not 
begin  to  a  marked  extent  until  later,  when  what  remains  of 
the  food  passes  down  into  the  large  intestine.  Ordinarily  the 
small  intestine  is  devoid  of  disagreeable  odor,  showing  the 
absence  of  putrefactive  changes. 

It  is  evident  therefore  that  the  chemical  nature  of  the  food 
is  a  factor  of  great  importance  in  determining  the  character 
of  the  complex  reactions  which  follow  the  real  digestive  proc- 
esses in  the  upper  part  of  the  intestine.  Here  we  have  nor- 
mally the  work  of  enzymes,  and  this  is  always  followed  by 
bacterial  destruction  of  what  is  left.  But  we  must  distinguish 
between  fermentation  changes  and  putrefactive  changes,  the 
former  being  characteristic  of  carbohydrate  food  and  the  lat- 
ter of  protein  food.  As  one  or  the  other  of  these  pre- 
dominates, the  chemical  processes  taking  place  must  vary. 
Throughout  the  length  of  the  small  intestine,  and  in  the  be- 
ginning of  the  large  intestine  active  absorption  takes  place, 
but  between  the  enzymes  and  the  bacteria  a  struggle  for  the 
possession  of  the  field  is  in  progress  all  the  time.  Theoret- 
14 


194  PHYSIOLOGICAL    CHEMISTRY. 

ically.  without  the  bacteria,  the  foods  would  undergo  complete 
digestion  and  be  practically  all  absorbed,  but  before  this  ideal 
condition  can  be  reached  the  parasitic  bacteria  begin  their  work 
and  rob  the  body  of  part  of  its  food. 

Acid  Fermentation.  Just  when  this  competition  on  the 
part  of  the  acid-producing  bacteria  begins  is  hard  •  to  say. 
Through  the  upper  third  of  the  small  intestine  the  reactions 
are  essentially  those  of  true  pancreatic  digestion,  and  there  is 
at  no  time  a  sharp  line  of  demarkation  between  this  zone  and 
the  following  one.  The  point  in  the  intestine  where  the  acid 
fermentations  begin  is  a  fluctuating  one  and  must  vary  with 
the  time  which  has  elapsed  since  the  beginning  of  the  digestion 
as  well  as  with  the  character  of  the  food.  The  enzymic  and 
acid  fermentation  zones  must  besides  overlap  each  other;  that 
is,  in  the  central  part  of  the  tract  the  two  kinds  of  changes 
must  go  on  simultaneously.  Lactic  and  butyric  fermentations 
are  favored  by  a  nearly  neutral  medium,  and  this  is  for  a  time 
secured  by  the  slow  neutralization  of  portions  of  these  acids 
formed  through  the  alkali  of  the  pancreatic,  the  intestinal  and 
the  bile  secretions.  As  the  foods  push  farther  down  the  neu- 
tralizing action  of  the  alkali  becomes  less  and  less  marked,  and 
finally  the  characteristic  acid  decomposition  becomes  the  prin- 
cipal feature. 

In  some  animals  this  acid  fermentation,  to  the  almost  com- 
plete exclusion  of  putrefactive  changes,  is  easily  recognized. 
The  food  of  the  herbivora  contains  an  excess  of  pentoses, 
starches  and  other  carbohydrates,  and  these  produce  sugar 
enough  to  furnish  a  large  portion  for  intestinal  fermentation. 
The  feces  of  these  animals  have  not  the  disagreeable  odors  of 
those  of  carnivorous  animals,  where  the  putrefactive  reactions 
are  very  marked  and  the  fermentations  of  very  minor  impor- 
tance. In  animals  with  a  mixed  diet  this  condition  can  be 
very  largely  changed  at  will  by  causing  a  variation  of  the  food 
given  them. 

With  the  disappearance  of  the  larger  part  of  the  carbohy- 
drates through  absorption  and  acid  fermentation,  the  products 


CHANGES    IN    INTESTINES.       THE    FECES.  IQS 

of  fermentation  being  themselves  partly  absorbed,  the  activity 
of  the  putrefactive  organisms  gains  the  upper  hand  and  large 
numbers  of  complex  reactions  follow.  The  nature  of  some  of 
the  bodies  produced  in  this  way  has  been  already  referred  to 
and  further  facts  may  now  be  given.  In  laboratory  experi- 
ments on  pancreatic  digestion  it  will  be  recalled  that  two  gen- 
eral results  are  obtainable.  In  working  with  the  pancreas  or 
pancreatic  extract  plus  fibrin  or  casein  we  add  thymol  or  chlor- 
oform water  if  it  is  desired  to  secure  the  maximum  enzymic 
effect,  but  if,  on  the  contrary,  the  bacterial  as  well  as  the  en- 
zymic decompositions  are  desired  this  protective  addition  is 
omitted  and  putrefaction  soon  becomes  apparent.  In  the  ani- 
mal body  the  acid  fermentation  products  take  the  place  in  a 
measure  of  the  chloroform  or  other  substances  used  in  the 
laboratory  experiments.  Indol  and  skatol  have  been  already 
referred  to  as  characteristic  disintegration  products  resulting 
from  the  action  of  bacteria  on  proteins ;  there  are  many  others 
in  addition  to  these,  and  most  of  them  are  compounds  of  the 
aromatic  group.  Under  the  conditions  of  their  appearance  in 
the  intestines  these  disintegration  residues  must  be  largely 
formed  from  the  albumoses,  peptones,  leucine  and  t3Tosine  of 
the  previous  enzymic  digestions;  in  comparatively  rare  cases 
it  is  possible  that  the  putrefaction  may  take  place  with  portions 
of  left-over  original  proteins  which  for  some  reason  escaped 
digestion  proper.  Phenol,  paracresol,  phenylacetic  acid,  phe- 
nylpropionic  acid,  para-oxyphenylacetic  acid,  glycocoll,  methyl 
mercaptan,  hydrogen  sulphide,  marsh  gas  and  still  other  sub- 
stances, including  various  volatile  fatty  acids  and  carbon  diox- 
ide, have  been  found  here  along  with  the  indol  and  skatol. 
These  various  products  are  produced  mainly  in  the  large  intes- 
tine, and  here  again  we  find  certain  limitations  to  the  extent 
of  the  reactions.  Through  the  small  intestine  the  contents 
have  remained  very  soft  and  liquid,  but  in  the  large  intestine 
normally  a  marked  absorption  of  water  takes  place,  from 
which  the  contents  become  thick  and  at  times  almost  hard. 
This   loss   of  water  interferes   greatly  with   the   progress   of 


196  PHYSIOLOGICAL    CHEMISTRY. 

putrefaction.  In  addition  to  this  the  work  of  the  bacteria  is 
hindered  by  the  accumulation  of  the  products  of  their  own 
production.  Some  of  these  products  have  to  a  certain  degree 
a  bactericidal  action  and  tend  to  check  the  more  rapid  bacterial 
development.  It  follows  therefore  that  when  the  rectum  is 
reached  in  the  downward  progress  of  the  intestinal  contents 
there  may  still  be  present  remains  of  putrescible  matters  which 
might  have  been  broken  down  if  all  the  conditions  had  been 
favorable. 

This  brings  us  to  a  consideration  of  the  final  remains  or 
the  feces,  but  first  a  word  must  be  said  about  the  absorption  of 
certain  products  in  the  lower  stretches  of  the  intestine.  Not 
only  are  the  normal  digestive  products  taken  up  from  both 
intestines,  from  the  small  intestine  mainly,  but  various  prod- 
ucts of  the  bacterial  reactions  referred  to  follow  the  same 
course.  The  importance  of  this  fact  is  very  great  from  two 
directions  at  least.  The  excessive  production  of  such  a  body 
as  indol  is  always  a  consequence  of  increased  bacterial  activity 
which  is  often  a  pathological  phenomenon.  The  indol  may 
escape  partly  with  the  feces,  but  a  portion  is  always  absorbed 
and  is  oxidized  in  the  tissues,  the  liver  probably,  from  which 
it  passes  into  the  blood  and  later  into  the  urine,  where  it  is 
recognized  in  the  form  of  indican.  The  amount  of  indican 
and  certain  similar  substances  detected  in  the  urine  is  a  meas- 
ure then  of  the  extent  of  putrefactive  changes  going  on  in  the 
intestine.  In  this  oxidation  an  atom  of  oxygen  is  taken  up 
and  indoxyl  is  formed : 

CsHtN  +  O  =  CsHeCOH)  N. 

This  indoxyl,  like  other  basic  substances,  always  finds  sul- 
phuric acid  to  combine  with  to  yield  indoxyl  sulphuric  acid 
or  a  salt  of  the  form 

CsHeN\ 

or  indican. 

Phenol  is  another  product  of  intestinal  putrefaction  and  in 
part  passes  also  into  the  circulation  from  the  lower  intestme 


CHANGES    IN    INTESTINES.       THE    FECES.  19/ 

to  reach  the  urine  finally  in  the  form  of  ethereal  sulphate. 
Other  substances  referred  to  above  as  putrefactive  products 
follow  the  same  course ;  in  part  they  escape  with  the  feces,  and 
in  part  they  suffer  absorption  to  be  more  or  less  changed  and 
finally  eliminated  by  the  urine.  The  importance  of  the  two 
substances  for  our  purpose  is  mainly  diagnostic.  They  are 
not  absorbed  in  sufficient  quantity  to  be  poisonous,  but  if  found 
easily  in  the  urine  this  points  to  a  more  than  normal  intestinal 
disintegration  of  protein  substances  or  their  derivatives.  If 
the  lower  intestine  becomes  for  any  reason  clogged  with  fecal 
products  which  prevent  the  easy  downward  passage  and  escape 
of  the  contents  of  the  small  intestine,  time  is  given  for  the 
more  prolonged  action  of  the  bacteria,  resulting  in  the  accumu- 
lation of  these  disintegration  products.  In  nearly  all  condi- 
tions of  high  fever  the  same  thing  is  observed.  The  urine 
test  is  frequently  therefore  a  suggestion  of  an  approaching 
pathological  condition,  or  of  an  aggravated  condition. 

In  another  direction  these  bacterial  products  have  interest 
and  importance.  While  the  traces  of  indol,  phenol,  etc.  found 
may  be  quite  harmless,  it  does  not  necessarily  follow  that  other 
things  produced  in  the  same  way  may  be  equally  harmless. 
On  the  contrary,  some  of  the  putrefactive  products  found  in 
the  intestine  are  violent  poisons  and  their  absorption  consti- 
tutes an  element  of  danger  to  the  body  as  a  whole.  In  labo- 
ratory experiments  it  is  an  extremely  simple  matter  to  obtain 
from  certain  bacterial  cultures  soluble  products  which  are  very 
toxic.  These  are  the  toxins  formed  by  the  bacteria  and  when 
injected  into  the  circulation  of  animals  are  capable  of  produc- 
ing poisonous  effects.  Similar  bodies  are  undoubtedly  formed 
in  the  intestines  if  the  bacteria  there  present  become  excessive 
in  number.  Sometimes  the  microorganisms  themselves  pene- 
trate the  intestinal  walls  and  pass  to  other  parts  of  the  system, 
being  collected  finally  by  the  urine.  But  the  peculiar  poisons 
produced  by  them  are  much  more  likely  to  be  absorbed  into 
the  circulation  and  give  rise  to  special  symptoms  at  points  far 
removed  from  the  infected  intestine.     No  one  of  these  intes- 


I9<^  PHYSIOLOGICAL    CHEMISTRY. 

tinal  toxins  has  been  isolated  and  definitely  recognized,  but 
with  them  other  bodies  are  formed  which  are  readily  detected 
in  the  urine,  as  is  the  indican  referred  to  above.  In  the  urine 
of  typhoid  fever,  and  of  other  pathological  conditions  also,  cer- 
tain complex  aromatic  products  are  always  present  which  give 
rise  to  the  well-known  reaction  designated  as  the  diazo  reac- 
tion of  Ehrlich.  When  a  mixture  of  weak  solutions  of  sodium 
nitrite  and  sulphanilic  acid  is  added  to  this  pathological  urine 
under  certain  conditions  it  strikes  a  carmine  to  garnet  red 
color,  due  to  the  formation  of  an  azo  compound  of  some  kind. 
The  urine  must  add  an  aromatic  body,  different  from  those 
normally  present,  to  aid  in  the  formation  of  this  azo  coloring 
substance.  With  normal  urine  an  orange  color  is  usually 
obtained,  but  this  deeper  red  is  characteristic  of  some  bacterial 
product  probably,  of  the  exact  nature  of  which  we  are  still 
in  ignorance. 

THE  FECES. 

Composition.  In  the  lower  intestine  the  absorption  of 
water  is  one  of  the  most  important  of  the  changes  taking  place 
and  this  leaves  what  remains  in  a  semi-solid  condition  ready 
for  final  discharge.  The  amount  of  water  left  in  the  feces  is 
quite  variable,  and  although  the  fecal  mass  may  appear  hard 
the  water  content  is  usually  70  to  85  per  cent.  In  the  thinner 
pathological  discharges  it  may  be  much  higher. 

At  first  sight  it  might  appear  that  the  feces  should  consist 
mainly  of  undigested  residues,  and  this  was  long  held  to  be 
the  case ;  but  we  now  know  that  such  substances  may  make  up 
the  least  important  part  of  the  discharge.  The  several  kinds 
of  products  present  may  be  roughly  divided  as  follows : 

1.  Bacteria. 

2.  Products  formed  by  bacteria. 

3.  Remains  of  the  digestive  ferments. 

4.  Epithelium  and  mucus  from  the  intestinal  walls. 

5.  Food  residues  partly  or  wholly  undigested. 

In  the  normal  fecal  discharge  all  these  groups  are  repre- 
sented, but  incidentally  there  may  be  many  other  things  pres- 


CHANGES    IN    INTESTINES.       THE    FECES.  1 99 

ent.  There  are  often  substances  which  become  accidentally 
mixed  with  the  food  and  which  are  not  attacked  by  the  diges- 
tive secretions.  There  may  be  remains  of  various  substances 
taken  into  the  body  as  remedies,  for  example  oxide  or  sulphide 
of  bismuth  from  bismuth  subnitrate,  or  chalk  or  other  insolu- 
ble substance  taken  in  the  same  way. 

NORMAL   FECES. 

For  comparison  it  is  necessary  to  have  something  as  a 
standard,  and  as  such  a  fecal  discharge  from  a  condition  ap- 
proaching starvation  might  be  taken.  In  such  feces  there  are 
no  food  residues,  but  the  other  things  are  abundantly  repre- 
sented. Many  analyses  of  feces  have  been  made  from  per- 
sons who  for  a  period  of  several  days  had  consumed  no  food 
and  these  give  some  idea  of  the  character  of  the  discharges 
which  might  be  expected  when  the  minimum  of  food  is  con- 
sumed and  no  more.  It  has  been  calculated  in  this  way  that 
about  lo  to  12  grams  daily  is  the  average  normal  discharge 
from  a  man  of  70  kilograms  weight,  due  to  other  sources  than 
the  remains  of  food.  Numerous  attempts  have  been  made  to 
find  the  average  composition  of  feces  from  a  diet  which  con- 
tains just  enough  protein,  fat  and  carbohydrate  to  keep  the 
body  in  normal  condition.  Some  of  the  results  are  given  in 
the  table  below,  in  per  cent. 

Water.  Dry  Subst.  Fat.  Nitrogen. 

Mixed  diet   76.S  23.5  6.2  i.o 

Mixed  diet   85.0  IS-O  4-0  0.9 

Milk  diet    71.2  28.8  4-8  1.4 

Milk   diet    77-0  23.0  2.7  0.9 

The  moist  feces  in  the  adult  may  weigh  from  50  grams 
about  to  400  or  500  grams  daily  in  health.  The  average 
weight  is  about  150  gm.  The  variations  depend  on  the  indi- 
vidual and  also  largely  on  the  character  of  the  food.  This 
last  is  illustrated  in  the  following  table  from  Kdnig's  "  Nahr- 
ungsmittel,"  where  for  certain  foods  the  daily  consumption 
is  given  and  also  the  weight  of  the  moist  and  dry  feces  in 
grams. 


-OO  PHYSIOLOGICAL    CHEMISTRY. 

Food  and  Amoint,      Grams.  Feces,  Gkams. 

Fresh.  Dr>-.  Fresh.  Dr>'. 

Roast  beef   884  366.8  65.3  17.7 

Eggs,  boiled  948.1  247.4  42.7  13.0 

^lilk    2438.0  315.0  96.3  24.8 

^lilk    4100.1  529.7  174.0  50.0 

Milk    2291.0  296.0  )  - 

Cheese    200.0  123.8  \  ~^ 

Cheese    517  320.0  )  ^"^  ^ 

Meat  614  135.9  i 

Bread    450  303-3,^  299.1  46.5 

Bacon    95.6  \ 

Cornmeal    (mush).  750  641.4  198.0  49.3 

Potatoes    ...3077.6  819.3  635.0  93.8 

Rice    638.0  551.9  194.6  27.2 

Flour  (as  bread)  . .  500  438.8  95.2  23.5 

Carrots     2566  351.6  1092.6  85.1 

Peas    959.8  835.6  927.1  124.0 

It  will  be  noticed  here  that  the  highest  weight  of  feces  cor- 
responds to  the  hig-h  weights  of  certain  vegetable  foods  which 
are  rich  in  cellulose.  Meat  and  milk  in  proper  amount  yield 
feces  which  are  not  excessive,  but  with  milk  and  cheese  in 
excessive  amounts  the  weight  of  feces  becomes  large. 

In  what  may  be  called  normal  feces  certain  relations  exist 
between  the  nitrogen,  the  fat  and  the  ash.  if  we  understand 
by  the  term  "  normal  feces  "  a  product  containing  no  excess 
of  unabsorbed  food,  as  just  explained.  Such  feces  contain  as 
nitrogen  compounds  only  those  substances  that  are  left  over 
from  the  digestive  secretions  or  bacterial  ferments,  or  are  pro- 
duced from  the  intestines  themselves,  while  the  "  fats  "  are 
ether-soluble  products  of  similar  origin  rather  than  the  orig- 
inal complex  glycerides.  In  some  cases  recently  reported  the 
following  figures  were  obtained  which  will  serve  as  illustra- 
tions. Three  dogs  of  similar  character  were  fed  on  a  meat 
diet  through  a  number  of  days,  two  receiving  just  enough  to 
keep  them  in  nitrogen  equilibrium,  while  the  third  received  an 
excess  of  meat.  The  results  of  analyses  of  the  feces  were, 
from  the  dried  substance,  in  per  cent  amounts  as  follows : 


CHANGES    IN    INTESTINES.       THE    FECES.  20I 


Dog. 

N. 

Fat. 

Ash. 

I 

8.S9 

13.18 

19.24 

II 

8.8s 

11.46 

22.09 

III 

10.56 

10.12 

14.14 

The  high  nitrogen  of  No.  Ill  indicates  an  excess  of  pro- 
tein ;  this  being  high,  the  fat  and  ash  must  be  correspondingly 
low.  The  difference  in  the  two  kinds  of  feces  becomes  more 
apparent  when  it  is  remembered  that  a  much  higher  factor 
must  be  used  in  multiplying  the  N  values  to  obtain  original 
substance  in  III  than  is  probable  for  I  and  II.  Since  in  III 
an  excess  of  protein  is  known  to  be  present  the  factor  ap- 
proaches 6.25,  while  for  I  and  II  it  is  probably  not  over  4.5 
or  5.0.  Something  will  be  said  below  about  the  general  char- 
acter of  the  important  fat  and  nitrogen  substances  present  in 
the  feces. 

THE  ANALYSIS  OF  FECES  AND  INTERPRETATION  OF  RESULTS. 

This  may  extend  to  the  recognition  of  a  large  number  of 
products,  but  usually  includes  the  detection  and  determination 
of  a  few  important  ones  only.  Fat  of  some  kind  is  always 
present,  hence  a  qualitative  test  is  of  little  value.  The  total 
amount  of  fat  must  be  determined  by  some  kind  of  an  extrac- 
tive process.  Nitrogen  is  determined  generally  by  the  Kjel- 
dahl  method  and  special  tests  are  made  for  proteins  or  their 
more  immediate  derivatives.  Occasionally  unchanged  starch 
and  other  carbohydrates  are  present  which  may  be  recognized 
by  methods  given  below.  The  amount  and  character  of  the 
ash  is  sometimes  of  value,  likewise  the  reaction.  Below  a  few 
details  will  be  given  about  some  of  these  tests. 

Separation.  Ordinarily  tests  are  made  on  feces  of  mixed 
diets,  but  frequently  it  is  desirable  to  observe  the  character  of 
feces  following  special  diet  and  not  modified  by  the  product 
from  a  previous  or  later  diet.  To  separate  the  feces  for  such 
tests  several  schemes  have  been  proposed.  It  is  best  to  give 
some  inert  substance  at  the  beginning  of  the  period  which  will 
pass  through  the  stomach  and  intestines  unchanged,  and  at 
the  conclusion  of  the  period  of  dieting  the  same  substance  may 


202  PHYSIOLOGICAL    CHEMISTRY. 

be  given.  Fine  precipitated  or  floated  silica,  powdered  char- 
coal, carmine  and  other  things  have  been  used  in  this  way. 
The  feces  to  be  examined  are  collected  between  the  discharges 
of  the  inert  and  insoluble  limiting  substances.  \^arious  de- 
vices are  also  used  to  collect  the  feces  apart  from  the  urine, 
which  is  essential  for  exact  tests. 

Reaction.  In  health  the  reaction  of  feces  with  litmus  is 
practically  neutral  in  most  cases,  but  at  times  either  acid  or 
alkaline  reaction  may  be  found.  \\'ith  excessive  putrefaction 
in  the  lower  intestine  various  aromatic  products  and  ammo- 
niacal  compounds  are  formed  which  may  show  alkaline  be- 
havior with  indicators  sensitive  in  this  direction.  On  the 
other  hand  free  fatty  acids  may  occasionally  be  present  in  suf- 
ficient quantity  to  give  a  distinct  acid  reaction.  In  speaking 
of  the  reaction  in  the  intestine  the  conditions  for  the  formation 
of  light  fatty  acids  were  explained.  Putrefaction  on  the  one 
hand  and  fermentation  on  the  other  are  the  important  factors 
in  this  connection;  and  these  depend  in  turn  largely  on  the 
diet.  Meat  diet  gives  usually  neutral  or  alkaline  reaction; 
with  excessive  carbohydrates  the  reaction  may  turn  to  acid. 
^^'ith  infants  on  mother's  milk  the  reaction  is  commonly  acid, 
while  with  cow's  milk  it  is  neutral  or  alkaline.  These  tests- 
refer  to  the  behavior  with  litmus,  but  with  phenol-phthalein, 
which  is  not  very  sensitive  with  weak  alkalies,  an  acid  reac- 
tion due  to  carbonic  acid  is  often  obtained.  This  fact  should 
be  kept  in  mind,  since  the  question  of  reaction  is  often  a  ques- 
tion of  indicators.  ^ 

In  testing  for  the  reaction  some  of  the  mixed  feces  is  spread 
on  one  side  of  the  test  paper  by  means  of  a  glass  rod;  the  color 
effect  is  looked  for  on  the  other  side  of  the  paper.  If  the  feces 
are  not  quite  moist  it  will  be  necessary  to  rub  up  with  a  little 
water. 

Diy  Residue  or  Solids.  As  explained  above,  the  larger  part  of  the  fecal 
discharge  is  always  water.  The  amount  of  solid  matter  is  best  obtained 
by  drying  a  weighed  portion,  at  a  relatively  low  temperature,  in  a  current 
of  hydrogen  or  air.  B\-  evaporating  over  a  water-bath  there  will  be  al- 
ways some  loss  of  volatile  substances  besides  water.     It  is  verj'  difficult 


CHANGES    IN    INTESTINES.       THE    FECES.  203 

to  obtain  a  perfectly  dry  product  on  the  water-bath  in  most  cases,  espe- 
cially if  fat  is  present.  For  most  purposes  it  is  safest  to  evaporate  a 
relatively  large  amount  to  moderate  dryness  on  the  water-bath,  after  mix- 
ing the  weighed  feces  with  a  little  alcohol.  This  air-dry  product  is 
weighed  and  finely  powdered  and  a  new  portion  is  weighed  out  for  the 
final  complete  drying,  at  a  temperature  of  105°  in  the  air  bath.  There 
will  be  some  little  loss  by  volatilization  of  light  acids  and  other  sub- 
stances. 

For  this  kind  of  work  a  vacuum  drying  apparatus  which  can  be  heated 
to  a  moderate  temperature  renders  good  service.  It  is  also  possible, 
where  time  is  not  an  object,  to  finally  dry  the  air-dried  product  in  a 
desiccator  under  sulphuric  acid;  that  is,  in  the  forms  of  drying  apparatus 
in  which  the  acid  is  above  and  the  substance  below.  For  this  purpose 
the  air-dried  feces  must  be  thoroughly  powdered,  or  distributed  in  a  very 
thin  layer.  In  some  pathological  stools  there  is  an  abundance  of  fat,  even 
to  one-half  of  the  total  solids.  In  such  cases  a  perfect  drying  is  always 
difficult  with  any  process. 

Specific  Gravity.  An  exact  determination  of  this  datum 
is  not  easily  made,  as  the  occluded  gases  interfere  greatly  with 
the  test.  The  normal  specific  gravity  is  about  1.045  to  1.070, 
but  may  be  much  lower  pathologically.  Fatty  stools  may  have 
a  specific  gravity  as  low  as  0.935. 

THE  TOTAL  FATS. 

In  the  analysis  of  feces  a  number  of  substances  are  included 

under  the  term  "  fat."     In  the  extraction  of  dried  feces  with 

some  solvent  everything  which  goes  into  solution  is  classed 

as  crude  fat,  to  be  more  fully  identified  by  special  tests  later. 

Besides  the  fats  proper  feces  may  contain  fatty  acids  and  their 

soaps,  traces  of  lecithin,  cholesterol,  cholalic  acid  and  other 

bodies  soluble  in  ether  or  chloroform.     In  the  acidified  feces 

these  substances  go  into  solution,  the  acids  of  the  soaps  being 

taken  up  also. 

For  this  extraction  it  is  customary  to  add  enough  acid  to  impart  a  faint 
acid  reaction  to  the  feces  and  then  evaporate  to  dryness  with  addition  of 
sand  or  other  inert  insoluble  substance.  The  dry  residue  may  be  trans- 
ferred to  a  paper  tube  and  extracted  with  anhydrous  ether  in  the  Soxhlet 
apparatus.  A  better  plan  is  to  spread  a  weighed  portion  of  the  mixed 
and  acidified  feces  over  paper  such  as  is  employed  in  the  well-known  milk 
fat  extraction  process.  The  test  is  completed  by  drying  the  paper  and 
extracting  in  the   Soxhlet  tube,  as  in  the  case  of  milk.     The  results  so 


204  PHYSIOLOGICAL    CHEMISTRY. 

obtained  are  higher  than  tliose  from  the  ordinary  process  and  the  time 
required  for  extraction  much  shorter.  But  it  is  not  easy  to  obtain  in  this 
way  enough  fat  for  further  study,  as  not  much  more  than  lo  gm.  can  be 
easily  worked. 

The  amount  of  crude  fat  in  tlie  dry  feces  is  variable  but  may 
make  up  in  tlie  mean  about  25  per  cent.  Much  of  it  under 
normal  conditions  must  be  derived  from  other  sources  than  the 
unutilized  original  fat  of  the  foods ;  a  portion  is  always  de- 
rived from  residues  from  some  of  the  intestinal  secretions,  and 
from  organized  elements  thrown  off  from  the  walls  of  the  in- 
testines. The  extent  of  the  utilization  of  the  food  fat  depends 
largely  on  its  physical  character,  especially  its  melting  point. 
The  solid  fats  with  high  melting  point  are  but  poorly  utilized 
as  the  following  figures  illustrate,  in  which  the  amount  of 
loss  in  the  feces  from  different  kinds  of  fat  is  given,  (v. 
Noorden.) 

>felts.  Loss. 

OHve  oil    liquid  2.3  per  cent. 

Goose  fat    25°  2.5 

Lard    34  2.5 

Bacon    43  2.6 

Mutton  tallow   49  7.4 

Stearin  plus  almond  oil 55  10.6 

Pure  stearin    (and  palmitin) 60  90.0 

The  free  fatty  acids  do  not  appear  to  be  as  well  absorbed  as 
are  the  neutral  fats,  and  in  general  from  mixed  vegetable 
foods  the  fat  loss  in  the  feces  is  much  greater  than  from  the 
animal  foods. 

Pathologically  there  may  be  a  very  great  increase  of  fat  in 
the  feces,  so  great  in  fact  as  to  be  readily  recognizable  by  the 
eye.  This  is  especially  true  in  cases  where  the  flow  of  bile 
into  the  intestine  is  diminished  or  altogether  hindered.  The 
fat  in  the  dry  feces  may  then  amount  to  50  per  cent  of  the 
whole.  Any  derangement  of  the  normal  pancreatic  functions 
leads  also  usually  to  an  increase  of  fat  in  the  feces.  In  the 
last  case  protein  and  carbohydrates  would  suffer  also  in 
absorption. 


CHANGES    IN    INTESTINES.       THE    FECES.  205 

ANALYSIS  OF  THE  CRUDE  FECES  FAT. 
The  method  of  separating  or  extracting  the  crude  fat  has 
been  briefly  referred  to  above.  An  extract  obtained  in  this 
way  may  be  used  for  a  number  of  tests  after  having  been 
weighed.  The  recognition  of  all  the  substances  in  it  is  prac- 
tically out  of  the  question,  but  there  is  no  difficulty  in  making 
an  approximate  separation  if  enough  fat  be  used.  A  simple 
heating  test  will  show  the  presence  of  light  and  volatile  fats, 
which,  however,  are  not  usually  present  in  more  than  traces. 
The  following  scheme  will  be  sufficient  for  the  recognition  of 
the  more  important  constituents.  The  extraction  is  completed 
in  the  Soxhlet  apparatus,  the  ether  distilled  off  and  the  residue 
dried  and  weighed. 

Cholesterol.  Cholesterol  is  not  a  fat  chemically  and  therefore  does  not 
undergo  saponification.  This  behavior  makes  it  possible  to  recognize  it. 
Add  to  the  crude  fat  some  alcoholic  potassium  hydroxide,  for  i  gm.  of 
fat  about  1.5  gm.  of  the  stick  alkali  in  25  cc.  of  alcohol.  Boil  under  a 
reflux  condenser  half  an  hour  and  then  drive  off  the  alcohol.  From  the 
dry  residue  extract  the  unchanged  cholesterol  by  use  of  an  excess  of 
ether.  The  substance  is  rather  slowly  soluble  and  a  little  soap  may  be 
dissolved  at  the  same  time.  The  result  is  fairly  accurate.  The  ethereal 
solution  of  the  cholesterol  is  evaporated  and  the  residue  weighed.  By 
evaporating  a  little  of  an  ethereal  solution  of  the  substance  on  a  glass 
slide  a  residue  is  secured  which  will  serve  for  microscopic  identification. 
Cholesterol  crystaUizes  in  large  thin  plates. 

Cholalic  Acid.  Cholalic  acid  from  the  bile  is  an  important  constituent 
of  the  crude  fat,  provided  this  is  obtained  from  slightly  acidified  feces. 
This  acid  may  be  detected  in  the  soap  left  after  extraction  of  the 
cholesterol  as  just  explained.  The  soap  is  mixed  with  a  little  water  and 
acidified  with  dilute  sulphuric  acid  to  free  all  the  organic  acids.  The 
mixture  is  extracted  with  ether  in  a  separatory  funnel.  After  completed 
extraction  the  ether  is  evaporated,  leaving  the  free  fatty  and  other  acids. 
To  these  a  slight  excess  of  barium  hydroxide  solution  is  added  and  heat 
applied  to  form  barium  soaps.  While  warming,  the  mixture  must  be  well 
stirred  or  shaken.  The  separation  to  be  made  depends  on  the  fact  that 
the  barium  soap  of  cholalic  acid  is  soluble  in  about  25  parts  of  hot  water 
while  the  true  fatty  soaps  are  not.  Therefore  on  washing  with  plenty  of 
hot  .water  the  bile  acid  soap  dissolves.  By  evaporating  the  solution  to  a 
small  volume,  acidifying  with  dilute  sulphuric  acid  and  shaking  with  ether 
the  cholalic  acid  will  pass  again  into  ethereal  solution,  from  which  it  may 
be  recovered  on  evaporation.  The  acid  may  be  recognized  by  mixing  with 
strong   sulphuric   acid.      A   yellow  solution   results    which    soon    shows    a 


206  PHYSIOLOGICAL    CHEMISTRY. 

green  fluorescence.  The  acid  may  be  identified  also  by  mixing  with  a 
small  amount  of  water  and  a  little  cane  sugar,  and  adding  then  a  few 
drops  of  strong  sulphuric  acid.  A  red  color  develops  which  becomes 
purple.  The  sulphuric  acid  must  be  added  in  just  sufficient  quantity  to 
warm  the  mixture  to  about  70°  or  75°  C.  This  test  is  the  Pettenkofer  bile 
test. 

Fatty  Acids  Proper.  After  separating  the  cholesterol  and  cholalic  acid 
as  just  described  the  true  fatty  acids  are  left  in  the  form  of  insoluble 
barium  soaps.  By  acidifying  with  a  little  hydrochloric  acid  and  shaking 
with  ether  these  acids  go  into  solution  and  may  be  recovered  by  evapora- 
tion of  the  ether.  The  fatty  acids  of  any  lecithin  originally  present  are  in- 
cluded, as  the  lecithin  would  be  decomposed  in  the  first  saponification.  The 
acids  of  soaps  as  well  as  of  neutral  fats  are  also  included  if  the  original 
extraction  was  made,  as  assumed,  on  acidified  feces. 

Lecithin.  The  separation  of  lecithin  as  such  from  feces  is  not  practicable 
but  the  amount  max-  be  estimated  from  the  phosphoric  acid  separated  in 
the  saponification.  In  the  above  tests  the  glycerophosphoric  acid  would  go 
as  barium  salt  along  with  cholalic  acid  into  the  hot  water  solution.  The 
phosphate  could  be  recognized  or  determined  in  this.  But  it  may  be 
estimated  much  more  accurately  by  using  some  of  the  original  crude  fat. 
This  is  mixed  with  some  sodium  carbonate  and  ashed  carefully.  Then  a 
little  saltpeter  is  added  to  complete  oxidation ;  the  fused  mass  is  dissolved 
in  water.  The  phosphoric  acid  may  be  determined  by  titration  with 
uranium  nitrate,  or,  better,  with  molybdic  acid  by  the  Pemberton  method. 
In  this  way  it  is  usually  possible  to  secure  enough  phosphate  to  make  an 
accurate  titration,  in  most  cases,  by  starting  with  a  gram  of  crude  fat. 

Soaps.  The  above  tests  give  the  total  acids.  It  may  be  desirable  to 
measure  the  amount  present  in  the  form  of  soaps.  For  this  purpose  a 
double  extraction  is  necessary.  In  one  case  the  feces  are  dried  and  ex- 
tracted with  ether  without  preliminary  acid  treatment.  The  soaps,  not 
being  ether  soluble,  remain  behind.  Then  a  second  extraction,  after  acidi- 
fication, is  made;  the  result  gives  the  total  fats  and  acids  and  the  differ- 
ence between  the  two  extractions  shows  the  acid  due  to  soaps. 

For  these  extractions  the  paper  coil  method  is  very  satisfactory  as 
sufficient  extract  may  be  obtained  from  about  10  gm.  of  moist  feces  for 
satisfactory  weighing. 

CARBOHYDRATES. 

Under  this  term  starch,  sugar,  gums  and  cellulose  must  be 
included.  With  a  vegetable  diet  the  last  named  is  always 
present,  while  on  a  purely  animal  diet  not  one  of  the  group 
can  be  found  in  the  feces. 

Starch.  This  substance  is  found  commonly  in  feces,  under 
normal  as  well  as  pathological  conditions,  and  especially  when 
the  diet  has  contained  starch  in  the  form  of  coarse  meal,  which 


CHANGES    IN    INTESTINES.       THE    FECES.  20/ 

is  difficult  of  digestion.  This  is  readily  shown  with  cornmeal 
and  other  products  containing  much  cellulose.  On  the  other 
hand  with  fine,  well-sifted  flours  in  which  the  starch  granules 
are  easily  turned  into^  paste  by  boiling  or  baking,  the  utiliza- 
tion of  the  starch  is  usually  much  more  complete.  For  the 
identification  of  starch  several  methods  have  been  applied, 
some  direct,  others  indirect. 

The  recognition  of  starch  by  the  microscope  usually  fails,  as  the  outline 
of  the  granules  is  destroyed  by  the  process  of  cooking.  When  the  cooking 
has  been  imperfect,  however,  the  granules  may  be  intact  and  in  a  condition 
suitable  for  identification.  The  iodine  test  is  frequently  satisfactory. 
For  this  the  feces  are  boiled  with  water  and  filtered.  In  the  filtrate  the 
iodine  solution  is  added  as  for  other  tests.  To  facilitate  the  separation  of 
the  starch  from  cell  structures  a  little  hydrochloric  acid  may  be  added  to 
the  boiling  water. 

The  amotmt  of  starch  is  best  determined  after  conversion  into  sugar. 
This  may  be  accomplished  by  prolonged  heating  with  a  little  hydrochloric 
acid,  which  changes  the  starch  into  glucose.  This  may  be  measured  by 
one  of  the  copper  reduction  processes,  but  not  without  some  difficulty  as 
the  solution  is  always  highly  colored.  The  necessary  precautions  can  not 
be  given  here. 

The  normal  starch  content  of  the  feces  is  always  small,  but 
in  disorders  of  digestion,  especially  with  diminished  activity 
of  the  pancreas,  more  starch  may  be  found.  In  very  young 
children  the  consumption  of  starchy  foods  is  very  often  fol- 
lowed by  the  appearance  of  starch  in  the  feces. 

Sugar.  The  presence  of  traces  of  sugar  in  the  normal  feces 
has  been  frequently  affirmed  and  as  often  denied.  The  larger 
part  of  the  more  recent  evidence  on  the  question  goes  to  show 
that  sugar  is  not  normally  present  even  in  traces.  All  forms 
of  sugar  are  very  soluble  and  easily  absorbed  from  the  intes- 
tine. The  ability  of  sugar  to  escape  absorption  through  the 
whole  length  of  the  intestinal  tract  would  therefore  appear 
very  problematical.  Even  in  disease  sugar  is  of  rare  occur- 
rence in  the  feces  as  far  as  has  been  determined  by  experiment. 
But  the  detection  of  traces  is  not  an  easy  task. 

Pathologically  sugar  seems  to  pass  through  the  intestine  and 
escape  with  the  feces  only  when  the  conditions  for  absorption 


20S  PHYSIOLOGICAL    CHEMISTRY. 

through  the  intestinal  walls  are  reversed.  This  is  the  case  in 
diarrhoea  because  of  the  more  rapid  movement  downward  in 
the  intestine  and  because  of  the  diminished  or  interrupted  flow 
from  the  intestine  to  the  blood.  There  may  be  an  increased 
loss  of  proteins  at  the  same  time. 

The  detection  of  traces  of  sugar  calls  for  a  preliminary  extraction,  and 
purification  of  the  extract  from  substances  which  might  interfere  with  the 
copper  or  analogous  tests.  A  fermentation  test  is  sometimes  made  and 
without  preliminarj-  treatment.  This  depends  on  the  spontaneous  decom- 
position of  the  sugar  by  bacteria  with  liberation  of  gas  which  is  collected 
and  measured.  Starch  present  gives  the  same  result,  however,  but  not 
so  rapidly. 

Cellulose.  This  is  a  common  constituent  of  feces  after  the 
consumption  of  vegetable  foods.  Practically  no  digestion  of 
cellulose  takes  place  in  the  small  intestine  of  man,  but  in  the 
large  intestine  there  is  sometimes  a  bacterial  destruction.  The 
detection  of  cellulose  is  not  difficult,  although  the  methods  are 
somewhat  complicated.  The  best  of  the  methods  depend  on 
the  solution  of  other  carbohydrates  by  treatment  with  weak 
acid  and  then  with  alkali  and  finally  with  water.  The  mixture 
is  filtered  and  the  residue  washed  with  alcohol  and  ether;  it  is 
crude  cellulose  contaminated  with  a  little  ash  and  protein  sub- 
stance, both  of  which  may  be  determined.  The  appearance 
of  cellulose  in  the  feces  has  of  course  no  pathological  sig- 
nificance. 

Gums.  As  these  are  not  common  articles  of  food  they  do 
not  occur  usually  in  the  feces.  AMien  they  are  consumed  in 
pastry  and  confectionery  they  may  be  found  later  in  the  feces 
since  they  are  not  digested  with  readiness  in  many  cases. 
Some  of  the  gums  are  but  slightly  soluble  and  undergo  pan- 
creatic digestion  slowly.  Experiments  ha\-e  shown  that  gum 
tragacanth  and  gum  arabic  may  be  found  in  considerable 
quantity  after  their  consumption  in  bon-bons. 

NITROGEN  AND  THE  PROTEINS. 

Nitrogen  is  found  in  the  feces  in  many  combinations. 
Some  of  these  represent  residues  from  the  digestive  operations 


CHANGES    IN    INTESTINES.       THE    FECES.  209 

and  some  are  found  in  secondary  products  formed  by  bacterial 
or  chemical  action.  Some  of  the  molecular  combinations  are 
large,  while  others  are  relatively  small.  No  conclusion,  there- 
fore, as  to  the  weight  of  the  nitrogenous  bodies  can  be  drawn 
from  the  nitrogen  found,  but  the  datum  has  value  from  other 
standpoints. 

Total  Nitrogen.  The  total  nitrogen  in  the  feces  may  be  accurately  de- 
termined by  a  combustion  process,  but  most  readily  by  the  Kjeldahl  proc- 
ess which  is  now  everywhere  employed.  This  depends  on  the  conversion 
of  the  nitrogen  into  ammonia  by  prolonged  heating  with  sulphuric  acid  to 
which  a  very  little  metallic  mercury  is  added.  Often  a  mixture  of  pure 
sulphuric  acid  and  potassium  sulphate  is  employed.  At  the  end  of  the 
digestion  the  mixture  is  made  alkaline  with  a  slight  excess  of  ammonia- 
free  sodium  hydroxide  and  distilled.  The  ammonia  formed  is  collected  in 
standard  acid  and  measured  in  this  by  titration  of  the  excess  of  acid  with 
standard  alkali. 

Even  in  condition  approaching  starvation,  when  no  food 
proteins  can  possibly  be  present,  the  feces  always  show  some 
nitrogen,  which,  as  pointed  out  above,  must  come  from  the 
secretions  thrown  into  the  intestines  and  from  the  remains  of 
bacteria  and  their  products.  A  part  of  this  nitrogen  there- 
fore has  once  been  absorbed  to  be  later  thrown  back  into  the 
intestine,  which  fact  must  be  kept  in  mind  in  making  deduc- 
tions from  the  nitrogen  found  as  to  the  loss  of  nitrogen  in 
assimilation.  Although  usually  overlooked  the  nitrogen  ex- 
isting in  the  bacterial  cells  is  an  appreciable  quantity  and  often 
makes  up  a  good  fraction  of  the  whole.  It  has  recently  been 
shown  that  in  normal  feces  nearly  one-third  of  the  dry  weight 
may  often  be  made  up  of  the  bacteria. 

The  amount  of  nitrogen  excreted  increases  with  the  food 
consumed  in  general,  and  especially  if  this  food  contains  a 
large  amount  of  indigestible  substance.  The  nitrogen  of  a 
meat  diet  is  always  more  completely  utilized  than  is  the  nitro- 
gen of  beans,  for  example,  where  there  is  considerable  cellu- 
lose to  disintegrate.  The  nitrogen  in  this  case  is  largely  in 
the  form  of  protein  residues,  and  may  be  detected  as  will  be 
pointed  out  below.  In  pathological  conditions  of  the  diges- 
ts 


210  PHYSIOLOGICAL    CHEMISTRY. 

tive  tract  there  may  be  a  j^^reat  increase  of  unutilized  nitrogen. 
This  is  more  especially  true  of  failures  in  the  pancreatic  diges- 
tion than  it  is  of  failure  in  the  work  of  the  stomach. 

Proteins.  The  most  important  question  to  consider  here  is 
that  of  proteins  themselves  in  the  feces.  Nitrogen  in  other 
forms  has  a  far  different  meaning  since  it  may  represent  bodies 
which  have  been  already  digested  and  absorbed,  and  then 
thrown  into  the  intestine  again.  But  nitrogen  as  protein  rep- 
resents practically  waste  in  most  cases.  Among  the  protein 
substances  which  may  be  found  sometimes  in  feces  these  may 
be  mentioned :  albumins  proper,  casein,  nucleo-proteids,  albu- 
moses.  peptones  and  naturally  more  or  less  of  certain  albu- 
minoid bodies  which  are  digested  with  difficulty.  The  certain 
detection  of  all  these  bodies  under  all  conditions  is  not  always 
possible  with  our  present  knowledge.  Some  of  the  simplest 
of  the  tests  employed  will  be  briefly  mentioned.  The  soluble 
substances  only  are  considered  here. 

Albumins.  Acidify  the  fresh  feces  with  dihite  acetic  acid  and  extract 
with  distilled  water.  The  acid  prevents  casein  and  mucin  from  going 
into  solution  at  the  same  time.  Filter  through  good  Swedish  paper  and 
apply  tests  to  the  filtrate.  Albumose  and  peptone  go  into  solution  with 
this  treatment. 

Albumins  proper  are  coagulated  by  heating  the  filtrate,  while  the  de- 
rived proteins  do  not  respond  to  this  test.  The  albumins  give  also  the 
biuret  test  and  are  precipitated  by  solution  of  potassium  ferrocyanide  in 
presence  of  acetic  acid.  But  albumose,  not  peptone,  responds  to  the  same 
test. 

Albumoses  and  Peptones.  By  extracting  as  above,  coagulating  any  albu- 
min possibly  present  and  filtering,  the  filtrate  may  be  used  for  albumose 
and  peptone  tests.  In  the  filtrate  free  from  albumin  zinc  sulphate  or  am- 
monium sulphate  may  be  used  to  precipitate  albumose.  In  the  filtrate 
from  this  peptone  may  be  recognized  by  the  biuret  test. 

Casein.  This  is  sometimes  found  in  the  feces  of  children  on  a  milk 
diet.  To  recognize  it  these  tests  may  be  made.  The  fresh  feces  may  be 
extracted  first  with  rather  weak  sodium  chloride  solution  to  take  out  solu- 
ble albumins,  then  with  weak  acid  to  complete  the  removal  of  such  bodies. 
The  casein  may  next  be  brought  into  solution  with  sodium  hydroxide,  not 
too  strong,  and  obtained  in  the  filtrate.  In  such  a  filtrate  acetic  acid  pro- 
duces a  voluminous  precipitate  if  casein  is  present,  but  mucin  is  also  pre- 
cipitated. Casein,  however,  redissolves  in  an  excess  of  acetic  acid  while 
mucin   does  not.     After  filtering  the  casein  may  usually  be  thrown  out 


CHANGES    IN    INTESTINES.       THE    FECES.  2 1  I 

again  by  cautious  addition  of  alkali  to  the  neutral  point,  but  the  precipi- 
tate is  not  as  characteristic  as  in  the  first  instance. 

Mucin.  This  was  formerly  supposed  to  be  a  common  and  abundant 
product  in  normal  feces,  but  this  is  not  the  case.  Pathologically  mucin 
may  be  present  so  as  to  be  recognizable  by  the  eye.  A  good  chemical 
test  for  small  amounts  is  still  lacking. 

Nucleo-proteid.  By  extracting  normal  feces  with  lime  water  and  acidi- 
fying with  acetic  acid,  a  bulky  precipitate  is  obtained  usually,  which  was 
supposed  to  be  mucin.  It,  however,  contains  phosphorus  and  belongs  to 
the  proteid  group.  The  substance  is  a  normal  product  in  traces  and  can 
be  found  in  feces  following  a  diet  free  from  nucleins.  It  is  therefore 
likely  that  traces  of  this  protein  are  brought  into  the  intestines  from  the 
breaking  down  of  the  intestinal  walls.  Pathologically  much  more  may 
be  found,  but  without  having  a  distinct  diagnostic  indication. 

Of  all  the  protein  substances  mentioned,  the  casein,  if  it 
occurs  in  large  quantities  in  infants'  feces,  has  perhaps  the 
greatest  importance  as  pointing  to  imperfect  digestive  power. 
There  can  be  no  question,  of  course,  as  to  its  origin.  Serum 
or  egg  albumin  as  such  could  rarely  be  present  because  such 
proteins  are  ordinarily  consumed  in  the  coagulated  condition. 
When  the  tests  point  to  the  presence  of  a  true  soluble  albumin 
the  result  shows  probably  the  entrance  of  albumin  from  the 
blood  by  a  reversal  of  the  normal  osmotic  process.  It  must 
be  remembered,  however,  that  true  albumin  is  very  rarely 
found  in  the  feces.  Occasionally  a  reaction  due  tO'  presence 
of  pus  or  blood  may  be  obtained,  but  the  albumose  or  peptone 
reactions  are  much  more  frequent.  In  diarrhoea  stools,  for 
example,  where  insufficient  time  is  given  for  absorption,  these 
bodies  may  be  found. 

Insoluble  Proteins.  The  detection  of  coagulated  proteins 
and  of  partly  disintegrated  albuminoids  is  practically  impos- 
sible. Remains  of  muscle  fibers  or  other  complex  substances 
essentially  protein  may  sometimes  be  recognized  by  the  micro- 
scope, but  they  are  beyond  chemical  identification. 

OTHER  NORMAL  AND  ABNORMAL  SUBSTANCES. 

It  will  not  be  necessary  to  discuss  the  occurrence  of  the 
various  putrefactive  bodies  of  bacterial  origin  which  are  al- 
ways found  in  the  feces.     We  have  here  indol,  skatol,  various 


212  PHYSIOLOGICAL    CHEMISTRY. 

phenols  and  aromatic  acids.  Leucine  and  tyrosine  are  occa- 
sionally found,  but  their  presence  is  generally  pathological, 
if  in  quantity  more  than  traces. 

Among  products  of  distinctly  pathological  origin  blood  and 
pus  may  be  mentioned ;  both  yield  albumin  and  the  corpuscles 
of  each  may  frequently  be  recognized  by  the  microscope.  It 
is  also  possible  to  recognize  the  coloring  matter  of  the  blood 
by  the  spectroscope.  It  has  been  mentioned  that  cholalic  acid, 
a  derivative  of  the  two  characteristic  acids  of  the  bile,  may  be 
found  with  the  fats  of  the  feces.  The  bile  acids  themselves, 
glycocholic  and  taurocholic,  are  also  found ;  likewise  the  bile 
pigments  or  their  disintegration  products.  Most  of  the  bile 
coloring  matters  fail  to  be  reabsorbed  from  the  intestine  into 
which  they  are  discharged  and  must  be  excreted  therefore  by 
the  feces  and  only  in  small  part  by  the  urine. 


SECTION    III. 

THE   CHEMISTRY  OF   THE   BLOOD,  THE 

TISSUES   AND  SECRETIONS 

OF   THE   BODY. 

CHAPTER    XI. 

THE   BLOOD. 

How  Supplied.  The  conversion  of  food-stuffs  into  ab- 
sorbable products  has  been  discussed  in  the  chapters  of  the  last 
section.  It  must  be  shown  now  how  these  products  are  util- 
ized. Sooner  or  later,  by  absorption  from  the  stomach  or  the 
intestines,  mainly  from  the  latter  organs,  they  enter  the  blood 
stream  through  two  principal  channels,  the  portal  vein  and  the 
lacteal  lymph  vessels  leading  to  the  thoracic  duct.  Ordinarily 
the  amount  of  absorption  from  the  walls  of  the  stomach  is  not 
great ;  only  when  a  very  large  quantity  of  easily  digested  food 
is  present  in  this  organ  or  under  the  influence  of  special  stim- 
uli is  the  passage  of  digested  substances  into  the  circulation 
here  appreciable.  The  small  intestine  with  its  very  consider- 
able surface  gives  up  the  bulk  of  the  absorbable  products  to 
the  blood  or  lymph  stream. 

The  digested  fats  pass  essentially  into  the  minute  lymphatic 
vessels  known  as  the  lacteals.  At  the  time  of  digestion  the 
contents  of  these  vessels  is  filled  with  a  milky  fluid  termed 
chyle,  but  at  other  times  the  lymph  flowing  here  is  nearly  clear. 
Minute  capillary  vessels  leading  to  the  portal  vein  take  up  the 
larger  portions  of  the  carbohydrates  and  protein  bodies  from 
the  small  intestine  and  thus  convey  them  to  the  liver,  where 
a  number  of  important  changes  take  place,  the  most  pro- 
nounced being  the  conversion  of  the  sugar  more  or  less  per- 
fectly into  glycogen.     These  reactions  will  receive  attention 


214  PHYSIOLOGICAL    CHEMISTRY. 

later.  Beyond  the  liver  the  hepatic  veins  lead  to  the  general 
circulation.  In  this  general  way  the  nutriments  reach  the 
blood  which  is  the  main  channel  of  distribution,  but  this  fluid 
is  far  from  being  a  simple  solution  or  mixture  of  these  nutri- 
ments in  the  condition  in  which  they  leave  the  alimentary 
canal.  The  most  important  of  the  blood  constituents  are,  in 
fact,  chemically  quite  distinct  from  anything  produced  in  the 
course  of  digestion.  Certain  organs  of  the  body  have  the 
important  function  of  working  over  these  digestive  products 
and  converting  them  into  the  things  required  in  the  blood. 
To  do  this  several  synthetic  reactions  are  necessary ;  how  these 
are  carried  out  we  do  not  know,  and  in  some  cases  we  are 
ignorant  also  of  where  they  take  place.  In  what  follows  some 
of  the  main  facts  in  this  connection  will  be  given. 

COMPOSITION    OF    THE    BLOOD. 

Quantitative  Variations.  It  is  evident  that  only  an  aver- 
age composition  can  be  in  general  considered  since  the  fluid 
is  in  a  state  of  constant  change.  Soon  after  a  meal  certain 
constituents  would  naturally  be  found  increased,  and  after  a 
period  of  fasting  a  deficiency  in  the  same  would  follow.  From 
what  has  been  said  it  is  further  apparent  that  the  blood  of  the 
portal  vein  would  be  found  much  richer  in  some  substances 
than  that  of  the  hepatic  vein  or  the  arteries.  It  must  also  be 
remembered  that  the  blood  is  not  a  homogeneous  fluid  but  con- 
sists of  a  true  solution  in  which  are  suspended  certain  cell 
structures.  We  may  therefore  consider  the  average  composi- 
tion of  the  blood  as  a  whole,  or  of  the  corpuscles  on  the  one 
hand  and  the  fluid  portion  or  plasma  on  the  other.  The  spe- 
cific gravity  of  normal  blood  varies  between  1.05  and  1.07;  the 
average  specific  gravity  of  the  serum  is  about  1.03. 

Approximately  the  blood  makes  up  7  to  8  per  cent  of  the 
body  weight;  therefore  in  an  individual  weighing  70  kilo- 
grams the  blood  weight  would  be  4.9  to  5.6  kilograms.  Of 
this  blood  weight  about  60  per  cent  belongs  to  the  plasma  and 
40  per  cent  to  the  corpuscles.     Among  the  various  recorded 


THE    BLOOD.  21  5 

analyses  of  human  blood  as  a  whole  the  following  may  be 
taken  as  best  illustrating  the  mean  composition,  in  looo  parts. 

BLOOD    ANALYSES. 

Men.  Women. 

Mean  of  11  Mean  of  8 

Analyses  Analyses. 

Water    779  791 

Solids   221  209 

Fibrin  2.2  2.2 

Hemoglobin    134-5  121.7 

Albumin  and  globulin  76.0  76.0 

Cholesterol,  fat,  lecithin   1.6  1.6 

Salts  and  extractives  6.8  7.4 

The  individual  analyses  from  which  these  means  are  taken 
show  rather  wide  variations.  Some  more  recent  analyses 
made  in  Bunge's  laboratory  show  the  distribution  of  the  min- 
eral matters  and  may  be  quoted,  the  figures  here  given  refer- 
ring to  the  blood  as  a  whole. 

Man  of  Woman  of 

25  years.  30  years. 

Water 789  824 

Solids   211  176 

Fibrin  3.9  i-9 

Hemoglobin  and  albumins  199-5  164.8 

Salts    7-9  8.6 

The  salt  content  was  made  up  in  each  case  as  follows : 

Man.  Woman. 

Sodium  chloride   2.701  3-417 

Sodium  oxide    921  1.862  -j-  potassa 

Sodium  phosphate   457  .267 

Potassium  chloride    2.062  1-623 

Potassium  sulphate    205  .193 

Potassium  phosphate  1.202  .835 

Magnesium  phosphate 137 


Calcium  phosphate 193  j 

7-878  8:6l5 

These  figures  do  not  show  the  distribution  of  the  salts  be- 
tween the  plasma  and  corpuscles.  In  the  original  analyses 
from  which  they  are  calculated  by  far  the  larger  part  of  the 


2l6  PHYSIOLOGICAL    CHEMISTRY. 

sodium  salts  was  found  in  tlie  plasma,  while  the  potassium 
salts  were  found  largely  in  the  corpuscles.  The  calcium  and 
magniesium  salts  occur  mainly  in  the  plasma.  In  the  blood 
the  excess  of  alkali  shown  exists  probably  mainly  as  carbon- 
ate. All  analyses  seem  to  indicate  a  difference  between  the 
blood  of  men  and  women.  The  male  blood  is  richer  in  solids. 
The  female  blood  on  the  other  hand  appears  to  be  slightly 
richer  in  the  mineral  salts. 

Blood  is  characterized  particularly  by  the  peculiar  compound 
containing  iron  present,  known  as  hemoglobin.  Many  of  the 
tests  for  the  recognition  or  identification  of  blood  depend  on 
this  substance,  which  is  found  nowhere  else.  As  a  whole 
blood  is  distinguished  by  the  phenomenon  of  coagulation  which 
is  connected  with  the  fibrin  present.  Because  of  the  great  im- 
portance of  this  phenomenon  it  will  be  briefly  discussed  here; 
the  details  of  the  subject  belong  to  physiology  rather  than  to 
chemistry  and  are  not  yet  sufficiently  worked  out  for  clear 
elementary  presentation. 

FIBRIN  AND  THE  COAGULATION  OF  BLOOD. 

As  has  been  already  pointed  out  fibrin  is  the  product  result- 
ing from  a  certain  reaction  in  which  a  forerunner  or  parent 
substance  called  fibrinogen  is  concerned.  As  it  exists  in  the 
blood  vessels  normally  this  fibrinogen  is  soluble  and  stable,  but 
when  the  vessel  is  pierced  and  the  contents  allowed  to  come  in 
contact  with  the  air  the  soluble  fibrinogen  becomes  the  insolu- 
ble fibrin,  which  is  the  well-known  string}^  substance  described 
in  an  earlier  chapter.  A  great  deal  has  been  written  on  the 
subject  of  this  spontaneous  coagulation,  which  is  now  gener- 
ally believed  to  be  brought  about  by  the  action  of  a  peculiar 
ferment  formed  by  the  breaking  down  of  the  white  blood  cor- 
puscles. From  these  cells  it  appears  that  a  special  zymogen 
which  has  been  called  prothrombin  is  first  formed;  this  in  the 
presence  of  calcium  salts  yields  the  true  fibrin  ferment  or  en- 
zyme called  thronibiii.  It  may  be  easily  shown  that  the  addi- 
tion of  ammonium  oxalate  or  some  other  precipitant  of  cal- 


THE    BLOOD.  2  1/ 

cium  salts  to  freshly  drawn  blood  will  prevent  its  coagulation. 
It  was  formerly  held  that  the  calcium  compounds  enter  into  a 
chemical  combination  as  part  of  the  fibrin  molecule,  but  Ham- 
marsten's  researches  seem  to  show  clearly  that  the  part  of  the 
calcium  is  in  the  formation  of  the  ferment. 

In  this  coagulation  it  appears  that  a  portion  of  the  original 
fibrinogen  is  split  off,  yielding  a  product  known  as  fibrin- 
globulin,  which  remains  in  solution ;  that  is,  the  whole  of  the 
fibrinogen  does  not  coagulate  as  such.  The  coagulation  may 
be  prevented  or  greatly  retarded  by  addition  of  oxalates  as 
just  referred  to,  and  also  by  addition  of  several  other  foreign 
substances,  as  acids,  alkalies,  strong  solutions  of  alkali  salts, 
sugar,  gum,  albumose  solutions,  glycerol,  etc.  An  excess  of 
carbon  dioxide  delays  coagulation,  as  shown  by  the  slower 
coagulation  of  venous  blood.  Blood  collected  from  a  vein  in 
a  polished  vessel  of  porcelain  or  in  a  vessel  whose  sides  have 
been  covered  with  oil  or  vaseline  coagulates  slowly.  On  the 
other  hand  collecting  in  a  vessel  with  a  rough  surface  hastens 
coagulation,  as  does  any  mechanical  agitation.  It  has  been 
shown  that  a  polished  platinum  wire  may  be  passed  through 
a  vein  without  inducing  coagulation,  while  a  thread  in  the 
same  position  will  collect  a  layer  of  fibrin. 

The  various  observations  which  have  been  made,  while  not 
affording  a  full  answer  to  the  question  why  the  blood  does  not 
coagulate  spontaneously  in  the  living  veins  or  arteries,  sug- 
gest several  important  reasons  to  account  for  this  absence  of 
the  reaction.  One  of  the  factors  evidently  present  in  all  ordi- 
nary coagulations  is  contact  with  a  rough  foreign  substance. 
The  foreign  substance  need  not  be  larger  than  the  specks  of 
dust  which  blood  can  gather  from  the  air.  In  leaving  a  vein  or 
artery  blood  naturally  comes  in  contact  with  such  particles, 
and  these  serve  as  nuclei  for  the  beginning  of  coagulation; 
much  as  a  minute  dust  particle  may  be  sufficient  to  start  crys- 
tallization in  a  strong  solution  of  alum.  In  the  body  the  blood 
is  normally  in  contact  with  vessels  with  very  smooth  walls. 
If  such  a  vessel  be  ligatured  at  two  points  and  the  sac  thus 


2l8  PHYSIOLOGICAL    CHEMISTRY. 

formed  be  cut  out  it  will  be  found  that  the  contained  blood 
will  remain  fluid  some  hours  or  days  even.  This  shows  that 
contact  with  liz'iiig  walls  is  not  the  element  preventing  coagu- 
lation. 

Apparently  blood  exists  normally  in  a  very  peculiar  condi- 
tion of  equilibrium,  in  which  not  one  but  several  factors  are 
concerned.  The  same  may  be  said  of  the  equilibrium  of  many 
salt  solutions.  Changes  of  temperature,  the  addition  of  for- 
eign bodies  in  traces  even,  stirring,  pouring  from  one  vessel 
into  another,  or  contact  with  the  dust  particles  of  the  air  in 
the  one  case  as  in  the  other  may  induce  a  change.  In  the 
living  vessels  of  the  body  as  well  as  after  leaving  the  body 
the  equilibrium  may  be  destroyed  and  a  coagulation  take  place. 
This  is  illustrated  in  the  intravascular  clotting  after  wounds  in 
which  the  vessels  as  a  whole  may  not  be  impaired;  injury  to 
the  lining  endothelium  results  in  throwing  foreign  particles 
into  the  blood  stream  sufficient  to  induce  clotting  or  coagu- 
lation. 

EXPERIMENTAL   ILLUSTRATIONS. 

Some  of  the  simpler  phenomena  connected  with  the  coagu- 
lation of  blood  may  be  readily  shown  by  experiment. 

Ex.  Have  ready  two  test-tubes.  Pour  into  the  first  one  cc.  of  a  cold 
saturated  solution  of  sodium  sulphate,  the  other  is  left  clean  and  dry.  De- 
capitate a  rat  and  allow  two  cc.  of  the  escaping  blood  to  flow  into  the 
tube  containing  the  sodium  sulphate.  The  rest  of  the  blood  is  collected 
in  the  dry  tube.  In  a  very  few  minutes  coagulation  takes  place  in  the  lat- 
ter tube,  while  it  is  prevented  by  the  sodium  sulphate  in  the  former. 

Allow  both  tubes  to  stand  at  rest  a  day  or  two.  In  the  salted  tube  it 
will  be  noticed  that  most  of  the  corpuscles  have  settled  to  the  bottom,  leav- 
ing a  clear  and  lighter  colored  liquid,  while  in  the  other  tube  the  coagulum 
has  begun  to  shrink  into  a  smaller  mass,  from  which  droplets  of  yellowish 
serum  ooze.     The  corpuscles  in  this  remain  with  the  fibrin. 

Ex.  Collect  a  quantity  of  slaughter-house  blood  by  running  two  vol- 
umes of  the  latter  into  one  volume  of  saturated  solution  of  sodium  sul- 
phate. Shake  the  mixture  and  allow  it  to  stand  at  a  low  temperature  sev- 
eral days.  Coagulation  does  not  occur,  but  a  gradual  precipitation  of  the 
corpuscles  is  observed,  leaving  a  yellowish  liquid  known  as  salted  plasma, 
which  may  be  poured  off  and  used  for  various  experiments. 

Ex.  Pour  a  few  cc.  of  the  salted  plasma  into  a  test-tube  and  dilute  it 
with  several  times  its  volume  of  water.     On  slight  warming  of  the  mix- 


THE    BLOOD.  2I9 

ture,  coagulation  follows.  The  effect  of  the  sodium  sulphate  is  to  prevent 
coagulation.     In  this  case  dilution  favors  it. 

Ex.  Pour  some  fresh  blood  into  a  clean  vessel  and  stir  it  thoroughly 
with  a  glass  rod,  if  a  small  quantity  in  a  beaker  is  taken,  or  with  a  stick 
if  a  larger  volume,  as  of  slaughter-house  blood,  is  used.  The  fibrin  gradu- 
ally separates,  and  entangles  most  of  the  corpuscles.  Save  the  serum  for 
tests  to  be  explained  and  wash  the  crude  fibrin  thoroughly  under  running 
water  to  remove  the  corpuscles  and  coloring  matter.  The  well-washed 
fibrin  is  white  and  stringy.  Fibrin  so  prepared  is  employed  in  many  ex- 
periments, especially  in  illustrating  digestion  phenomena.  On  the  large 
scale  it  is  used  in  the  manufacture  of  peptone. 

Ex.  To  illustrate  the  ready  digestion  of  fresh  fibrin  use  about  half  a 
gram  with  10  cc.  of  0.25  per  cent  hydrochloric  acid.  Keep  the  mixture 
some  hours  at  40°  C.  The  fibrin  gradually  dissolves  to  form  acid  albumin, 
which  may  be  obtained  in  solution  by  filtering  from  any  undigested  residue. 
The  careful  addition  of  a  little  sodium  carbonate  solution  produces  a  pre- 
cipitation of  the  acid  albumin. 

BLOOD    TESTS. 

The  serum  left  after  separation  of  the  fibrin  by  stirring,  con- 
tains much  of  the  blood  coloring  matter  and  may  be  used  as 
well  as  the  fresh  blood  for  many  tests,  some  of  which  will  be 
illustrated  here. 

Ex.  GuAiAcuM  Test.  To  a  little  blood  solution  in  a  test-tube  add  some 
fresh  tincture  of  guaiacum  and  then  a  few  drops  of  an  ethereal  solution 
of  hydrogen  peroxide.  Shake  the  mixture  and  observe  that  the  precipi- 
tated resin  has  assumed  a  blue  color,  more  or  less  marked.  In  this  test 
turpentine  oil,  which  has  been  shaken  with  air  in  a  bottle,  or  which  has 
been  exposed  to  the  air,  can  be  used  instead  of  the  solution  of  peroxide. 
Hydrogen  peroxide  is  developed  by  the  action  of  oxygen  on  turpentine. 
In  this  test  the  hemoglobin  seems  to  act  as  a  carrier  of  oxygen  to  the 
resin.     The  oxidation  product  of  the  resin  is  blue. 

Ex.  Hydrogen  Peroxide  Test.  A  reaction  somewhat  similar  to  the 
above  in  principle  is  observed  on  mixing  2  cc.  of  the  blood  with  10  cc. 
of  the  commercial  hydrogen  peroxide  solution.  The  hemoglobin  brings 
about  the  decomposition  of  the  peroxide  with  liberation  of  oxygen,  which 
escapes,  producing  froth. 

Reaction  of  Blood.  The  normal  reaction  of  blood  is  alka- 
line, which  cannot  be  observed,  however,  in  the  usual  way 
because  of  the  marked  color  of  the  pigment.  It  may  be  readily 
seen  by  working  in  the  following  manner: 

Ex.  Prepare  some  small  smooth  plaster  of  Paris  surfaces  by  pouring 
the  well-known  plastic  mixture   of  plaster   of   Paris   and   water   on   glass 


220  PHYSIOLOGICAL    CHEMISTRY. 

plates  and  allow  it  to  harden  several  hours  at  least.  The  prepared  plates 
are  removed  from  the  glass  and  soaked  in  a  neutral  solution  of  litmus  and 
are  then  allowed  to  dry.  The  test  proper  can  now  be  made  by  putting  a 
few  drops  of  the  blood  on  the  smooth  plaster  surface  and  allowing  it  to 
remain  there  five  minutes.  It  is  then  washed  off  with  pure  water,  when 
it  will  be  found  that  the  part  of  the  plate  which  has  been  covered  by  the 
blood  has  become  blue  from  the  action  of  the  alkali  of  the  blood  on  the 
neutral  litmus. 

Ex.  Heat  Test.  Heat  the  solution  of  blood  until  it  is  near  the  boiling 
temperature  and  note  that  the  red  color  .is  largely  destroyed  and  that  a 
brownish  precipitate  forms  which  contains  albumin  and  decomposed  color- 
ing matter.  Add  now  a  small  amount  of  sodium  hydroxide  solution  and 
observe  that  the  precipitate  disappears  while  the  blood  solution  becomes 
red  again  by  reflected  light,  but  greenish  by  transmitted  light. 

Ex.  Hemin  Crystals.  When  acted  on  by  acids  or  strong  alkalies  hemo- 
globin of  blood  is  broken  up  into  globin  and  a  characteristic  compound 
called  hematin.  Hematin  in  turn  is  acted  upon  by  hydrochloric  acid  yield- 
ing the  hydrochloride,  hemin,  which  appears  in  crystalline  form.  From 
the  name  of  their  discoverer,  these  crystals  are  called  "Teichmann's  crys- 
tals." Their  appearance  constitutes  one  of  the  best  tests  we  have  for 
blood,  and  can  be  illustrated  by  the  following:  Evaporate  a  drop  of  blood 
on  a  slide,  add  two  or  three  drops  of  glacial  acetic  acid,  and  boil.  Put  on 
a  cover  glass  and  allow  to  cool.  Minute  (microscopic)  plates  or  prisms 
separate  out.  If  old  blood,  a  stain,  for  instance,  is  examined,  it  is  neces- 
sary to  add  a  small  crystal  of  sodium  chloride  to  the  acetic  acid,  by  which 
means  sufficient  hydrochloric  acid  is  liberated  for  the  test.  The  crystals 
have  a  dark  brown  color  and  are  very  characteristic.  The  usual  forms  as 
found  in  human  and  other  blood  are  shown  below. 


Fig.    9.     Hemin    crystals,     i    is    from    human  Fig.  10.     Hemin  crys- 

blood  ;  2  from  seal  ;  3  from  a  calf ;  4  from  a  pig ;  tals    from    stains    of   hu- 

5  from  a  lamb;  6  from  a  pike;  7  from  a  rabbit,  man    blood.     (Landois.) 

(L.XNDOIS.) 


THE    BLOOD. 


221 


The  most  certain  means  of  identifying  blood,  however,  de- 
pends on  the  pecuHar  behavior  of  hemoglobin  toward  light, 
which  will  be  shortly  explained. 

HEMOGLOBIN. 

Composition.  In  the  systematic  classification  of  the  pro- 
tein bodies  hemoglobin  is  grouped  among  the  proteids  or  com- 
pound substances,  inasmuch  as  it  may  readily  be  broken  up 
into  a  fraction  containing  iron  called  hematin,  and  a  histone 
substance  called  globin.  This  cleavage  is  very  easily  effected 
by  the  action  of  weak  acids  and  in  the  mean  the  hematin  frac- 
tion is  found  to  amount  to  about  4.3  per  cent.  In  some  ex- 
periments as  much  as  94  per  cent  of  globin  has  been  recovered. 
It  is  therefore  likely  that  only  the  two  substances  are  present. 
The  properties  of  hemoglobin  are  not  quite  constant,  inasmuch 
as  from  different  bloods  products  of  slightly  different  compo- 
sition have  been  obtained.  It  is  possible  to  secure  the  hemo- 
globin in  crystalline  condition  suitable  for  analysis.  A  num- 
ber of  such  determinations  have  been  made  and  from  them 
formulas  have  been  calculated.  These  formulas  can  be  at 
best  only  more  or  less  close  approximations,  but  they  are  inter- 
esting as  illustrating  the  great  molecular  weights  here  con- 
cerned. 

Analyses  of  Hemoglobin.  Several  results  obtained  by 
different  observers  are  here  given.  The  variations  must  be 
partly  due  to  differences  in  methods  of  preparation  and 
analysis. 


c 

H 

N 

s 

Fe 

0 

Author. 

Horse. 

51-15 

6.76 

17.94 

0.39 

0-335 

23.42 

Zinnofsky. 

" 

54-40 

7.20 

17.61 

0.65 

0.47 

19.67 

Huefner. 

Dog. 

54-57 

7.22 

16.38 

O.S7 

0.336 

20.43 

Jaquet. 

" 

53-85 

7-32 

16.17 

0.39 

0.43 

21.84 

Hoppe-Seyler. 

Hen. 

52.47 

7.19 

16.45 

0.86 

0.335 

22.5 

Jaquet. 

In  the  first  analysis  the  ratio  of  the  sulphur  atoms  to  the 
iron  atoms  is  2  :  i ;  in  the  third  analysis  it  is  3:1.  On  the 
assumption  that  the  molecule  contains  but  one  atom  of  iron 


222  niYSIOLOGICAL    CHEMISTRY. 

the  minimum  molecular  weig-ht  which  may  be  calculated  from 

this  analysis  is : 

GssHmsNiBsOiisFeST 

It  is  interesting  to  note  that  the  molecular  weights  found 
in  this  way  are  practically  confirmed  by  the  determinations 
made  on  the  combining  power  of  hemoglobin  for  carbon  mon- 
oxide. Assuming  that  one  molecule  of  carbon  monoxide  is 
held  by  one  molecule  of  hemoglobin,  observations  of  the  vol- 
ume of  the  gas  absorbed  by  a  given  weight  of  the  blood  pig- 
ment lead  to  practically  the  same  result  as  was  obtained  by 
the  iron  method. 

Combinations  of  Hemoglobin.  The  great  importance  of 
hemoglobin  depends  on  its  power  of  forming  several  more  or 
less  stable  combinations  with  certain  gases.  Of  these  combi- 
nations that  wnth  oxygen  is  by  far  the  most  important ;  we 
distinguish  therefore  betw^een  hemoglobin  and  oxyhemoglobin. 
The  common  form  of  the  substance  is  really  the  latter,  al- 
though it  is  usually  referred  to  by  the  simple  term — hemo- 
globin. The  oxygen  of  oxyhemoglobin  is  very  loosely  held 
and  may  be  driven  out  from  its  union  by  the  aid  of  a  current 
of  other  gases,  or  by  the  pump.  The  amount  so  held  corre- 
sponds to  tW'O  atoms  of  oxygen  for  each  molecule  of  hemo- 
globin. This  oxygen  combining  powder  in  some  way  depends 
on  the  presence  of  the  iron  of  the  hematin. 

Oxyhemoglobin.  By  various  methods  this  substance  may 
be  obtained  in  crystalline  form,  the  crystals  being  often  2  mm. 
or  more  in  length.  From  blood  of  different  animals  different 
crystalline  forms  have  been  obsen^ed.  In  all  cases  the  crystals 
are  red  and  soluble  in  water;  they  are  more  easily  soluble  in 
water  containing  a  little  sodium  carbonate.  They  are  insolu- 
ble in  ether,  benzene  and  chloroform,  and  the  w-ater  solubility 
\-aries  with  the  nature  of  the  blood  from  which  they  were 
made.  That  from  the  blood  of  man  and  the  ox,  for  example, 
being  easily  soluble,  while  the  oxyhemoglobin  from  the  blood 
of  the  horse  or  dog  is  rather  slowly  soluble.  Because  of  their 
solubility  it  is  very  difficult  to  secure  crystals  from  human 
blood,  but  from  dog's  blood  they  may  be  made  as  follows : 


THE    BLOOD. 


Ex.  Beat  up  lOO  cc.  of  the  blood  thoroughly,  cool  to  a  low  temperature 
and  add  lo  cc.  of  ether  and  a  little  water.  Shake  this  mixture  thoroughly 
and  allow  it  to  stand  on  ice  over  night.  Filter  on  porous  paper,  squeeze 
out  the  mother-liquor  as  far  as  possible,  dissolve  in  a  little  water,  filter 
again  and  to  the  new  filtrate  add  one-fourth  its  volume  of  alcohol,  mean- 
while stirring  constantly.  Allow  the  mixture  to  stand  to  crystallize.  An 
illustration  is  given  of  the  usual  forms. 

A  simple  test  may  be  also  made  by  mixing  a  drop  of  dog's  blood  with  a 
drop  of  water  on  a  slide  and  allowing  it  to  partly  evaporate.  A  cover 
glass  is  then  put  on  and  the  crystals  are  looked  for  with  the  microscope. 

The  conditions  of  com- 
bination between  hemo- 
globin and  oxygen  have 
been  studied  by  several 
authors.  It  has  been 
found  that  by  exhausting 
blood  under  the  air  pump 
the  greater  part  of  the 
oxygen  becomes  free.  It 
has  been  found  further 
that  I  gm.  of  hemoglobin 
may  be  made  to  take  up  or 
give  off  something  over 
1.3  cc.  of  oxygen.  This 
reduces  to  2  atoms  of  oxy- 
gen for  I  atom  of  iron 
present  in  the  hemoglobin. 
Various  chemical  agents 
have  the  same  effect.  In 
the  case  of  certain  solu- 
tions the  action  is  a  chem- 
ical one,  while  with  sev- 
eral inert  gases  the  action  is  physical. 

These  reactions  are  accompanied  by  a  change  of  color  in  the 
oxyhemoglobin  or  blood  solution  experimented  upon.  Oxy- 
hemoglobin solutions  show  a  brighter  red  color  than  do  those 
containing  the  reduced  hemoglobin.  This  difference  is  well 
illustrated  in  the  contrasting  shades  of  arterial  and  venous 


Fig.  II.  Crystals  of  hemoglobin,  a  and 
b  from  human  blood ;  c  from  the  cat ;  d 
from  the  guinea-pig;  e  from  the  marmot; 
/  from  the  squirrel. 


224  PHYSIOLOGICAL    CHEMISTRY. 

blood,  the  former  containing  plenty  of  oxygen  in  combination 
while  the  latter  is  deficient.  The  loss  of  oxygen  is  illustrated 
by  some  simple  experiments : 

Ex.  Shake  about  lo  cc.  of  diluted  defibrinated  blood  with  a  few  drops 
of  ammonium  sulphide  solution  or  with  Stokes'  reagent.  (Stokes'  reagent 
is  a  solution  of  ferrous  sulphate,  to  which  a  small  amount  of  tartaric  acid 
has  been  added,  and  then  ammonia  enough  to  give  an  alkaline  reaction.) 
Warm  gently,  and  observe  that  the  bright  color  of  arterial  blood  gives 
place  to  the  darker  purple  of  venous.  On  shaking  the  mixture  now  with 
air  the  bright  red  color  returns.  For  the  success  of  this  experiment  where 
Stokes'  reagent  is  employed  it  should  be  freshly  prepared  before  use.  Vari- 
ous other  substances  behave  in  similar  manner. 

Ex.  Generate  some  hydrogen  gas  in  the  usual  manner,  and  allow  it  to 
bubble  through  diluted  defibrinated  blood.  A  change  of  color  follows  after 
a  time,  due  to  the  mechanical  loss  of  oxj'gen.  The  same  result  may  be 
accomplished  by  exhausting  the  oxygen  of  the  blood  by  means  of  an  air 
pump.     Exposure  to  the  air  restores  the  color  in  a  short  time,  as  before. 

The  color  change  may  be  noticed  readily  with  the  unaided 
eye,  but  is  much  more  marked  when  observed  in  the  spectro- 
scope, as  will  be  pointed  out  below. 

The  maximum  amount  of  oxygen  which  may  be  held  by 
the  hemoglobin  was  gi\'en  above  as  i  molecule  for  i  molecule 
of  the  pigment.  This  holds  only  for  strong  oxygen  pressure, 
however.  Under  lower  atmospheric  pressure  a  part  of  the 
oxygen  becomes  dissociated,  as  illustrated  by  these  figures 
given  by  Huefner  for  a  14  per  cent  hemoglobin  solution: 


Atmosphi 

:ric 

Per  cent  of 

Atmosph 

leric 

Per  cent  of 

Pressure  in 

Mg. 

Oxygen  Free. 

Bressure  in 

Mg. 

Oxygen  Free. 

760.0  mm. 

1.49 

238-5    1 

mm. 

4.60 

715-6 

1.58 

II9-3 

8.79 

620.8 

I.81 

47-7 

19.36 

524.8 

2.14 

23.8 

32.51 

477-1 

2-15 

4.8 

70.67 

357-8 

3-II 

The  loss  of  oxygen  does  not  become  marked  until  compara- 
tively low  pressures  are  reached. 

Carbon  Monoxide  Hemoglobin.  When  a  current  of  car- 
bon monoxide  is  led  into  a  blood  solution  it  displaces  the  oxy- 
gen and  forms  a  very  stable  compound.     One  molecule  of  the 


THE    BLOOD.  2  25 

monoxide  takes  the  place  of  the  molecule  of  oxygen  combined 
as  oxyhemoglobin.  This  reaction  is  accompanied  by  a  change 
of  color,  not  as  marked,  however,  as  the  change  from  reduced 
to  oxyhemoglobin.  The  combination  with  carbon  monoxide 
is  the  reaction  which  takes  place  in  cases  of  poisoning  with 
illuminating  gas,  which  contains  lo  to  25  per  cent  of  the  mon- 
oxide. The  addition  of  pure  air  does  not  displace  the  com- 
bined gas  except  where  a  great  excess  is  used. 

Ex.  Lead  a  current  of  illuminating  gas,  best  the  so-called  "  water  gas," 
into  50  cc.  of  blood  in  a  flask.  Continue  the  passage  of  the  gas  until  a 
distinct  cherry  red  color  is  produced.  When  the  combination  appears  to 
be  complete  treat  a  few  cc.  of  the  liquid  with  Stokes'  solution,  which  fails 
to  effect  a  reduction.  With  portions  of  the  mixture  further  tests  should 
be  made  to  illustrate  methods  of  differentiating  between  normal  blood  and 
blood  containing  much  monoxide.  The  differentiation  in  each  case  de- 
pends on  the  greater  stability  of  the  monoxide  hemoglobin  with  the  re- 
agent in  question. 

Ex.  Add  some  strong  solution  of  sodium  hydroxide  to  ordinary  blood. 
This  gives  a  brownish  green  precipitate  at  first  and  then  a  red  solution. 
Treat  blood  saturated  with  carbon  monoxide  in  the  same  manner.  This 
gives  a  red  precipitate  and  finally  a  red  solution. 

Ex.  Dilute  some  of  the  monoxide  blood  with  four  volumes  of  water 
and  to  the  mixture  add  an  equal  volume  of  a  3  per  cent  tannic  acid  solu- 
tion. The  red  color  persists  much  longer  than  it  would  in  the  case  of  a 
simple  oxyhemoglobin  solution,  which  should  be  tried  for  comparison. 

Ex.  To  about  half  a  cubic  centimeter  of  the  monoxide  blood  add  20 
cc.  of  water  and  10  drops  of  strong  yellow  ammonium  sulphide  solution. 
Shake  thoroughly  and  then  add  enough  dilute  acetic  acid  to  give  a  faint 
acid  reaction.  A  rose  red  color  appears,  while  with  normal  blood  decom- 
position products  are  formed  which  have  a  dirty  gray  color. 

Ex.  Mix  I  cc.  of  the  monoxide  blood  with  5  cc.  of  basic  lead  acetate 
solution  and  shake  well.  The  mixture  remains  red,  while  with  normal 
blood  under  the  same  conditions  a  brown  color  results. 

Experiments  have  been  carried  out  to  determine  what  por- 
tion of  the  hemoglobin  must  be  combined  with  monoxide  to 
have  death  follow.  It  appears  that  if  about  half  the  pigment 
in  the  blood  is  still  unchanged  recovery  may  be  expected  by 
free  use  of  air.  The  mere  action  of  a  great  excess  of  air  may 
gradually  displace  the  combined  monoxide.  The  spectro- 
scopic appearance  of  the  monoxide  hemoglobin  will  be  re- 
ferred to  below. 
16 


226  PHYSIOLOGICAL    CHEMISTRY. 

Nitric  Oxide  Hemoglobin.  Under  certain  conditions 
nitric  oxide.  NO.  may  be  combined  witli  bemog-lobin  to  form 
a  ver}^  stable  compound.  The  union  takes  place  molecule  with 
molecule  and  may  be  obtained  by  treating-  carbon  monoxide 
hemoglobin  with  the  nitric  oxide,  which  has  the  power  of 
breaking  up  the  monoxide  combination.  The  direct  action  of 
nitric  oxide  on  oxyhemoglobin  does  not  lead  to  the  desired 
result,  as  oxidation  of  the  NO  follows  and  the  acid  formed 
destroys  the  hemoglobin.  In  presence  of  urea,  however,  the 
direct  union  is  possible.  The  substance  forms  crystals  like 
those  of  oxyhemoglobin  and  gives  a  very  similar  spectrum. 

Sulphohemoglobin.  \\'hen  hydrogen  sulphide  is  led  into 
a  solution  of  oxyhemoglobin  in  presence  of  air  a  compound  is 
formed  which,  however,  is  not  permanent.  Decomposition 
soon  follows  and  a  greenish  brown  mixture  results.  \\'ith 
reduced  hemoglobin  away  from  the  air  a  true  compound  is 
formed  which  gives  a  characteristic  spectrum,  and  which  is 
much  more  stable. 

Other  Combinations.  Several  authors  have  described 
other  compounds  formed  by  the  union  of  hemoglobin  and 
gases.  Of  these,  so-called  carbohemoglobins,  acetylenehemo- 
globin  and  cyanhemoglobin  are  the  best  known.  These  com- 
binations are  but  slightly  stable  and  have  no  special  impor- 
tance. As  acetylene  was  formerly  prepared  on  the  laboratory 
scale  it  was  poisonous,  and  this  property  was  assumed  to  be 
due  to  its  action  on  hemoglobin,  which  w-as  thought  to  resem- 
ble that  of  carbon  monoxide.  Since  the  manufacture  of  acety- 
lene from  calcium  carbide  was  begun  this  notion  has  been  dis- 
pelled. The  action  of  acetylene  on  the  blood  is  very  weak. 
In  the  early  laboratory  product  impurities  present  were  prob- 
ably responsible  for  the  observed  effects. 

DERIVATIVES   OF  HEMOGLOBIN. 

Some  of  these  are  practically  identical  with  hemoglobin, 
while  others  are  products  of  complete  decomposition.  The 
first  to  be  considered  is : 


THE    BLOOD.  22/ 

Methemoglobin.  Oxyhemoglobin  in  solution  or  in  crystal 
form,  alone  or  in  presence  of  certain  reagents,  shows  a  great 
tendency  to  pass  OA^er  into  this  modification  which  contains 
just  as  much  oxygen  as  the  original,  held,  however,  in  stable 
combination.  Under  the  air  pump  methemoglobin  does  not 
give  up  any  oxygen,  while  from  oxyhemoglobin  nearly  the 
whole  of  the  extra  molecule  may  be  abstracted.  While  this 
formation  of  methemoglobin  takes  place  spontaneously  it  is 
greatly  hastened  by  the  action  of  several  substances,  some  of 
which  are  oxidizing  agents,  while  others  are  reducers.  Of  the 
oxidizing  substances  ozone,  potassium  permanganate,  potas- 
sium chlorate,  iodine,  potassium  ferricyanide  and  nitrates  have 
been  used,  while  such  reducing  agents  as  pyrogallol,  pyro- 
catechol,  hydroquinol  and  hydrogen  even,  acting  on  the  blood 
have  brought  about  a  formation  of  the  stable  methemoglobin. 
Certain  substances  given  as  remedies  have  the  power  of  con- 
verting the  oxyhemoglobin  into  methemoglobin.  Amyl  ni- 
trite, acetanilid  and  nitrobenzene  may  be  mentioned  here. 
The  poisonous  action  of  large  doses  of  potassium  chlorate  has 
long  been  supposed  to  be  due  in  part  to  the  same  reaction. 

Solutions  of  methemoglobin  are  not  bright  red  but  reddish 
brown,  and  the  crystalline  substance  is  also  brown.  The  color 
of  an  alkaline  solution  is  red,  but  this  is  not  due  to  a  recon- 
version into  oxyhemoglobin.  Certain  reducing  agents  have 
the  power  of  gradually  changing  the  methemoglobin  back  into 
oxyhemoglobin  and  then  into  reduced  hemoglobin.  Am- 
monium sulphide  and  Stokes'  reagent  work  in  this  way.  The 
conversion  may  be  followed  by  aid  of  the  spectroscope. 

A  product  known  as  acid  hemoglobin  is  formed  by  the  action 
of  w^eak  acids  on  hemoglobin.  This  appears  to  be  a  step  in 
the  formation  of  methemoglobin,  the  spectrum  of  which  it  re- 
sembles. With  strong  acids  decomposition  takes  place  and 
hematin  results. 

Hematin.  It  has  been  already  explained  that  hemoglobin 
breaks  up  readily  into  globin,  about  96  per  cent,  and  hematin, 
about  4  per  cent.     This  decomposition  follows,  as  just  men- 


228  PHYSIOLOGICAL    CHEMISTRY. 

tioned.  by  the  treatment  with  strong  acids,  and  also  by  various 
other  reactions.  The  product  from  reduced  hemoglobin  is 
known  as  hemochromogen,  while  from  oxyhemoglobin  oxy- 
hematin  or  common  hematin  is  obtained.  The  relations  may 
be  thus  illustrated : 

Hemoglobin  j  ^^°^'",  \   +  O  =  hematin 

I  hemochromogen  | .  _  p^  =  hematolin 

_  _  1  v.-     f  globin      1   -|-  HCl  =  hemin 

'^^  ^  I  hematin  .  —    O    =  hemochromogen 

(^  —   Fe  =:  hematoporphyrin 

Hematin  is  usually  obtained  as  an  acid  combination  or  ester. 
In  one  process  frequently  followed  blood  is  warmed  with  an 
excess  of  glacial  acetic  acid.  Crystals  containing  acetic  acid 
separate  on  cooling.  In  another  process  the  hydrochloride  is 
obtained ;  in  either  case  the  free  hematin  is  secured  by  saponi- 
fication with  weak  sodium  hydroxide  solution.  Several  for- 
mulas have  been  given  for  hematin ;  the  one  most  commonly 

accepted  is 

CssHssN^FeO., 

while  for  hemin  crystals  the  formula 

C3=H3oN4Fe03HCl 

has  been  given.  It  is  possible  that  different  analysts  have 
obtained,  not  the  same,  but  closely  related  products.  Hemin 
is  secured  in  minute  brownish  crystalline  plates,  hematin  as 
an  amorphous  bluish  black  insoluble  powder.  The  spectrum, 
which  is  important,  will  be  referred  to  later. 

Hematoporphyrin.  This  is  a  derivative  obtained  by  the 
action  of  acids  on  hematin.  In  this  treatment  the  iron  is  re- 
moved, as  illustrated  by  the  following  reaction,  when  hydro- 
bromic  acid  is  employed  as  the  decomposing  agent : 

C32H3.N4Fe04  +  2H,0  +  2HBr  =  2C>cHisN203  +  FeBr=  +  H^. 

The  substance  appears  to  be  related  to  and  isomeric  with  bili- 
rubin.    The  alkaline  solutions  of  hematoporphyrin  are  deep 


THE    BLOOD.  229 

red,  the  acid  solutions  incline  to  deep  violet  or  purple.     The 
acid  and  alkali  spectra  are  very  different  and  characteristic. 

The  relation  of  the  blood  coloring  matter  to  the  bile  pig- 
ments is  illustrated  by  these  formulas : 

G2H32N404Fe hematin 

C32H3eN406 hematoporphyrin 

C32H36N4O6 bilirubin 

C32H36N4OS biliYerdin 

Hematolin  is  the  name  given  to  an  iron  free  compound 
obtained  by  decomposing  hematin  in  absence  of  air. 

Hemochromogen.  This  is  obtained  by  reducing  a  hema- 
tin solution  with  ammonium  sulphide  or  with  zinc  dust  and 
alkali.  It  forms  a  dark  red  powder  insoluble  in  water  but 
soluble  in  alkalies.  The  solution  exposed  to  the  air  absorbs 
oxygen  and  appears  to  regenerate  hematin. 

Hematoidin  is  a  red-colored  pigment  which  has  been 
found  in  old  blood  extravasations.  It  seems  to  be  identical 
with  bilirubin. 

THE   OTHER  PROTEINS   OF  THE   BLOOD. 

In  addition  to  fibrin  and  hemoglobin  blood  contains  serum 
albumin  and  serum  globulin,  which  have  been  described  al- 
ready in  a  previous  chapter.  The  combined  substances  make 
up  about  7  per  cent.  They  may  be  approximately  separated 
by  precipitating  the  globulin  from  blood  serum  by  addition  of 
a  large  volume  of  water  and  also  by  salt  precipitation,  which 
may  be  illustrated  in  this  way : 

Ex.  Prepare  blood  serum  as  free  as  possible  from  corpuscles  as  already 
shown  and  mix  about  100  cc.  with  an  equal  volume  of  cold  saturated  am- 
monium sulphate  solution.  A  separation  of  the  globulin  follows.  Filter; 
the  filtrate  contains  practically  all  the  serum  albumin  which  may  be  coagu- 
lated by  boiling.  The  albumin  may  be  purified  by  long  dialysis.  To  rec- 
ognize the  globulin  in  the  precipitate,  first  wash  the  latter  with  more  half- 
saturated  ammonium  stilphate  and  then  dissolve  in  slight  excess  of  water. 
It  may  be  necessary  to  add  a  little  common  salt  to  assist  in  the  solution. 
On  heating  a  portion  of  this  solution  coagulation  follows.  On  diluting 
some  of  it  very  largely  with  water  precipitation  of  the  globulin  takes  place 
From  the  first  water  solution  most  of  the  salts  may  be  separated  by  long 
continued  dialysis. 


230  PHYSIOLOGICAL    CHEMISTRY. 

Magnesium  sulphate,  added  in  powder  form  to  saturation,  is  sometimes 
used  in  the  place  of  ammonium  sulphate  to  effect  the  separation  of  the 
albumin  and  globulin.  The  reaction  in  both  cases  depends  on  the  fact  that 
serum  albumin  may  be  salted  out  only  with  great  difficulty. 

It  is  an  interesting  fact  that  other  proteins  do  not  appear 
to  be  present  in  the  bl(3od.  The  various  proteins  consumed 
as  food  suffer  pecuhar  changes  somewhere  in  the  body  and  are 
converted  into  these  two.  These  in  turn  serve  for  the  prep- 
aration of  the  various  other  related  bodies  found  in  the  several 
tissues  of  the  organism.  Gelatin  may  be  formed  in  this  way 
from  the  proteins  of  the  blood,  but  it  does  not  appear  that 
gelatin  can  replace  other  proteins  as  a  food  since  it  is  deficient 
in  one  of  the  essential  protein  component  groups,  viz. :  the 
tyrosine  group. 

It  is  not  yet  known  how  constant  the  relation  of  serum  albu- 
min to  serum  globulin  is  or  whether  this  relation  is  the  same 
in  all  kinds  of  blood.  Egg  albumin  is  not  equivalent  to  serum 
albumin  physiologically,  since  if  injected  into  the  blood  it 
appears  soon  unchanged  in  the  urine.  The  albumins  of  re- 
lated animal  species  seem  to  be  nearly  alike,  but  this  does  not 
hold  absolutely  true  for  animals  of  w'idely  different  species. 

THE  SUGAR  OF  THE  BLOOD. 

This  is  found  in  the  plasma  and  has  generally  been  assumed 
to  be  glucose,  CcHjoOo,  although  good  reasons  may  be  as- 
signed for  the  assumption  of  other  sugars  as  well.  Ordi- 
narily the  simple  sugar  finally  formed  in  the  digestive  process 
is  glucose  and  the  possible  passage  of  other  sugars  into  the 
blood  has  commonly  been  overlooked.  As  the  amount  of 
sugar  in  the  blood  is  small,  about  0.15  per  cent  in  the  mean,  its 
certain  identification  is  a  matter  of  extreme  difficulty;  it  must 
be  remembered  that  separation  from  the  large  amounts  of  pro- 
teins present  must  be  complete  before  any  accurate  identifica- 
tion of  the  remaining  trace  of  sugar  may  be  thought  of.  The 
older  observers  depended  almost  solely  on  the  common  reduc- 
tion tests  which  are  not  very  sensitive  in  dealing  with  traces. 
Recent  investigators  have  shown  that  a  left-rotating  sugar  is 


THE    BLOOD.  23  I 

present  and  apparently,  also,  pentoses  in  traces.  As  glucose 
and  fructose  yield  the  same  osozone  this  simple  reaction  can- 
not be  applied  to  detect  a  fructose  content.  Occasionally  small 
amounts  of  disaccharides  appear  to  be  present.  Of  these  mal- 
tose passes  into  dextrose  by  inversion,  while  saccharose  and 
lactose  would  be  eliminated  as  such  by  the  kidney.  Gluco- 
ronic  acid  in  combination  is  also  present  and  this  may  be  con- 
founded with  a  sugar  in  some  of  the  tests.  More  will  be 
found  on  this  point  in  a  following  chapter. 

SALTS   OF  THE  BLOOD. 

The  total  mineral  matters  of  the  blood,  exclusive  of  the  iron 
of  the  hemoglobin,  amount  to  a  fraction  of  one  per  cent  only, 
but  still  are  of  very  considerable  importance.  These  salts  are 
largely  the  chlorides,  phosphates  and  carbonates  of  the  alkali 
metals,  the  potassium  salts  being  most  abundant  in  the  cor- 
puscles, while  the  sodium  salts  are  most  characteristic  of  the 
plasma.  It  is  believed  that  the  variations  in  this  salt  content 
are  very  small  normally.  The  nearly  constant  osmotic  pres- 
sure of  the  blood  points  to  this.  Slight  changes  are  speedily 
corrected  by  the  kidneys. 

Ex.  The  presence  of  reducing  carbohydrate  and  salts  in  the  blood  may 
be  demonstrated  in  this  way.  Mix  about  50  cc.  of  fresh  blood  with  300 
cc.  of  water  and  boil  vigorously  a  few  minutes.  A  drop  or  two  of  acetic 
acid  may  be  added  during  the  boiling  to  maintain  a  nearly  neutral  reac- 
tion. Filter  and  divide  the  filtrate,  which  should  be  perfectly  clear,  into 
two  parts.  Concentrate  one-half  to  a  volume  of  about  10  cc.  and  apply 
the  Fehling  test  for  sugar.  Concentrate  the  other  half  likewise  and  use 
portions  for  tests  for  phosphates  and  chlorides.  The  sulphate  test  usually 
fails  with  the  volume  of  blood  taken.  Evaporate  a  small  portion  of  this 
concentrate  nearly  to  dryness  on  a  glass  slide,  allow  what  is  left  to  cool 
and  crystallize.  Sodium  chloride  crystals  may  be  recognized  by  the  micro- 
scope. To  some  of  the  evaporated  residue  apply  the  flame  test  (with 
spectroscope)   for  potassium. 

GASES  OF  THE  BLOOD. 

The  blood  holds  several  gases  in  loose  combination.  These 
are  principally  oxygen,  nitrogen  and  carhon  dioxide.  Minute 
traces  of  argon  seem  to  be  present  also,  which  like  the  more 


2T,2  PHYSIOLOGICAL    CHEMISTRY. 

abundant  nitrogen  must  exist  in  a  condition  of  simple  solution. 
The  methods  of  accurate  gas  analysis  as  applied  to  blood  were 
developed  by  Lothar  Meyer,  who  made  a  number  of  deter- 
minations in  blood  from  different  sources.  These  methods 
have  been  further  improved  by  others  who  have  placed  many 
results  on  record. 

The  mean  amount  of  nitrogen  is  about  2  volume  per  cent. 
The  oxygen  and  carbon  dioxide  are  variable.  In  arterial 
blood  the  oxygen,  which  is  held  mainly  through  the  agency 
of  hemoglobin,  amounts  to  about  22  per  cent  by  volume;  that 
is.  from  too  cc.  of  arterial  blood  22  cc.  of  oxygen  in  the  mean 
may  be  obtained  by  aid  of  the  vacuum  pump.  The  venous 
blood  always  contains  less  oxygen,  probably  not  over  15  per 
cent  by  volume.  These  amounts  are  far  larger  than  could  be 
absorbed  from  the  air  through  the  partial  pressure  of  the  oxy- 
gen present.  In  fact  it  may  be  shown  that  only  a  fraction  of 
I  volume  per  cent  may  be  held  by  the  blood  plasma  perfectly 
free  from  corpuscles. 

The  loosely  combined  carbon  dioxide  may  vary  from  30  to 
40  volume  per  cent  in  the  arterial  blood,  while  in  venous  blood 
it  is  much  higher,  reaching  nearly  50  volume  per  cent  in  the 
mean.  This  gas  is  held  partly  in  the  form  of  bicarbonate  and 
partly  through  the  agency  of  the  proteins,  especially  the  hemo- 
globin. Most  of  the  carbon  dioxide  is,  however,  held  by  the 
serum  and  may  be  largely  drawn  out  by  aid  of  the  vacuum 
pump.  On  withdrawal  of  the  gas  other  weak  acid  bodies  are 
able  to  take  its  place  in  alkali  combination.  It  is  held  by  some 
observers  that  the  globulins  have  this  power.  In  acid  intoxi- 
cations where  mineral  or  organic  acids  increase  in  the  blood 
the  carbon  dioxide  rapidly  decreases  and  may  fall  to  a  tenth  or 
twentieth  even  of  its  usual  value.  These  stronger  acids  take 
the  alkali  and  there  is  therefore  nothing  left  to  hold  the  carbon 
dioxide. 

Some  of  these  points  will  be  taken  up  in  a  later  chapter  in 
discussing  respiration  phenomena. 

Other    Substances.      Besides    the    substances    mentioned 


THE    BLOOD.  233 

above  the  blood  always  contains  a  number  of  other  bodies  of 
more  or  less  importance.  Among-  these  may  be  mentioned 
the  fats,  soaps,  cholesterol,  lecithin  and  jecorin.  The  total 
fats  amount  ordinarily  to  about  0.2  per  cent,  but  after  a  meal 
may  be  temporarily  much  increased.  ]\Iinute  traces  of  fatty 
acids  as  soaps  may  be  also  present.  Cholesterol  appears  to  be 
present  in  free  form  in  traces  and  also  as  an  acid  combination 
or  ester.  Lecithins  are  present  in  very  small  amount  in  both 
corpuscles  and  plasma,  but  anything  like  a  quantitative  deter- 
mination does  not  appear  to  be  possible.  The  name  jecorin 
is  applied  to  a  peculiar  substance  containing  phosphorus,  de- 
scribed by  several  observers.  It  is  soluble  in  ether  like  lecithin 
and  seems  to  exist  in  combination  with  a  carbohydrate  group 
or  similar  reducing  residue.  The  substance  has  never  been 
obtained  in  form  pure  enough  for  analysis,  and  it  is  even  pos- 
sible that  it  may  be  a  mixture  of  several  compounds,  one  of 
which  is  a  combination  of  glucuronic  acid. 

Variations  in  Disease.  In  disease  the  normal  proportions  of  the  various 
substances  may  sufifer  marked  changes.  A  decrease  in  the  normal  num- 
ber of  corpuscles  (about  5  millions  to  the  cubic  millimeter  for  men,  4  to 
4.5  millions  for  women)  may  follow  to  the  extent  of  10  per  cent  or  more  in 
certain  anemic  conditions.  There  may  also  be  a  change  in  the  proportion 
of  hemoglobin  without  a  change  in  the  number  of  corpuscles.  The  meth- 
ods of  estimating  the  amount  of  hemoglobin  will  be  given  later.  The  salts 
in  the  blood  sufifer  a  percentage  decrease  after  consumption  of  large  quan- 
tities of  water,  but  only  temporarily.  An  actual  decrease  may  occur  in 
cholera  and  inflammatory  diseases.  The  normal  minute  amount  of  sugar 
is  increased  in  diabetes,  but  not  greatly,  because  of  the  eliminating  power 
of  the  kidneys.  It  may  be  temporarily  increased  by  the  use  of  certain 
drugs  such  as  curare,  amyl  nitrite,  chloral,  or  by  inhalation  of  chloroform 
vapor.  After  meals  rich  in  fats  there  is  a  temporary  increase  of  fat  in 
the  blood,  but  a  persistent  increase  is  noticed  in  the  blood  of  drunkards 
and  of  corpulent  individuals.  In  diseases  where  there  is  a  rapid  breaking 
down  of  proteins  there  is  usually  observed  an  increase  of  fat. 

A  loss  of  blood  to  the  extent  of  one-third  is  not  necessarily  dangerous 
if  it  be  withdrawn  slowly.  If  one-half  the  blood  is  lost  there  is  great 
danger  of  death.  Blood  may  be  added  by  transfusion,  but  for  safety  should 
be  from  an  animal  of  the  same  species.  The  serum  of  one  animal  has  usu- 
ally a  destructive  action  on  the  corpuscles  of  another.  Transfused  blood 
then  may  be  a  source  of  danger  rather  than  a  means  of  saving  life.  This 
peculiar  action  of  serum  will  be  referred  to  later  in  some  detail. 


CHAPTER    XII. 

THE  OPTICAL  PROPERTIES  OF  BLOOD.     USE  OF  THE 
SPECTROSCOPE  AND  OTHER  INSTRUMENTS. 

Solutions  of  hemoglobin  and  the  various  modifications  and 
derivatives  described  in  the  last  chapter  absorb  light  from 
certain  regions  of  the  spectrum.  The  character  and  extent 
of  this  absorption  are  such  as  to  afford  a  ready  means  of  iden- 
tifying blood  or  its  pigments  through  the  aid  of  the  spec- 
troscope. 

THE  SPECTRUM  FIELD. 

The  absorption  spectra  with  which  we  are  here  concerned 
are  all  found  in  the  middle  portions  of  the  spectrum  between 
the  Fraunhofer  lines  C  and  F,  that  is  in  a  region  easily  ob- 
served. For  practical  purposes  an  elaborate  instrument  is  not 
necessary.  Excellent  service  is  rendered  by  many  of  the 
smaller  direct  vision  spectroscopes.  For  quantitative  tests, 
howe\er,  much  more  complete  apparatus  is  required.  The 
blood  spectrum  differs  from  that  of  all  other  red  solutions  and 
is  very  easily  recognized  with  a  little  practice.  As  the  absorp- 
tive power  of  hemoglobin  is  very  great  dilute  solutions  only 
are  used  and  these  must  be  observed  in  a  rather  shallow  cell, 
preferably  in  one  with  parallel  sides  about  i  centimeter  apart. 
For  illumination  a  good  oil  lamp  flame  is  excellent;  a  steady 
gas  flame  may  also  be  employed. 

The  general  arrangement  of  the  essential  parts  of  the  spectroscope  are 
shown  by  the  following  figures.  Fig.  12  illustrates,  diagrammatically,  the 
path  of  the  light  rays  through  the  instrument.  From  the  source  F  the 
light  enters  the  collimator  tube  through  a  narrow  slit  and  reaches  the 
prism  P,  where  it  suffers  refraction  and  dispersion.  Beyond  the  prism 
the  rays  are  received  by  the  double  convex  lens  of  the  ocular  tube  and 
thrown  to  the  eyepiece  at  E.  A  magnified  virtual  image  is  formed  as 
shown  by  the  dotted  lines.  The  third  tube  carries  a  scale,  the  image  of 
which  is  reflected  into  the  ocular  and  shows  with  the  spectrum.  In  absorp- 
tion spectrum  analysis,  with  which  we  are  concerned  here,  the  light  at  F 

234 


OPTICAL    PROPERTIES    OF    BLOOD. 


235 


must  be  white  and  between  this  and  the  colhmator  sHt  a  cell  must  be 
placed  to  hold  the  colored  solution  or  diluted  blood.  This  is  shown  in 
the   next  figxire,  where  B   is   an  ordinary  kerosene   lamp   with  flat  wick. 


Fig.   12.     Diagram   of  simple  spectroscope. 


The  edge  of  the  flame  is  turned  toward  the  absorption  cell  and  slit.  The 
apparatus  here  figured  is  arranged  for  absorption  analysis  and,  with  parts 
to  be  described  later,  may  be  used  for  quantitative  work. 

For  most  simple  blood  examinations  the  small  direct  vision  spectroscope 
shown  below  may  be  used.     With  proper  combination  of  crown  and  flint 


Fig.    13.     Spectroscope   arranged  for  absorption   analysis. 

glass_  prisms  it  is  possible  to  practically  correct  the  refraction  and  leave 
a  field  with  satisfactory  dispersion. 

Variation  in  Spectra  by  Dilution.     In  all  dilutions  the 
positions  of  the  absorption  bands  remain  the  same,  but  their 


236  PHYSIOLOGICAL    CHEMISTRY. 

density  and  width  vary  with  the  concentration.  Tn  a  rela- 
tively strong  blood  solution,  for  example,  there  appears  to  be 
but  one  broad  oxyhemoglobin  band  between  D  and  E,  but  on 
proper  dilution  this  breaks  up  into  two  characteristic  bands. 
The  question  of  dilution  is  therefore  important  and  for  any 
given  instrument  and  light  the  observer  should  settle  this  by 
a  few  preliminary  experiments. 


Fig.    14.     Direct-vision    spectroscope. 

Spectrum  of  Oxyhemoglobin.  This  consists  of  two 
bands  in  the.  yellowish  green  portion  of  the  spectrum  between 
D  and  E.  The  bands  have  not  the  same  width,  the  one  near 
E  being  slightly  broader  than  the  other.  The  preparation  of 
proper  dilutions  may  be  illustrated  in  this  way : 

Ex.  Measure  out  5  cc.  of  defibrinated  blood  and  dilute  it  accurately 
with  120  cc.  of  water.  Filter  into  a  clean  flask  and  use  the  clear  filtrate 
for  tests  to  follow.    ]\Iark  this  mixture  Solution  No.  I. 

Dilute  50  cc.  of  No.  I  with  50  cc.  of  water  and  mark  the  mixture  Solu- 
tion No.  II. 

Dilute  50  cc.  of  No.  II  with  50  cc.  of  water  and  mark  the  mixture  Solu- 
tion No.  III. 

Dilute  50  cc.  of  No.  Ill  with  50  cc.  of  water  and  mark  the  mixture 
Solution  No.  IV. 

Dilute  50  cc.  of  No.  IV  with  50  cc.  of  water  and  mark  the  mixture 
Solution  No.  V. 

Dilute  50  cc.  of  No.  V  with  50  cc.  of  water  and  mark  the  mixture  Solu- 
tion No.  VI. 

Finally,  dilute  50  cc.  of  No.  VI  with  50  cc.  of  water  and  mark  the 
mixture  Solution  No.  VII. 

We  have  now  dilutions  beginning  with  i  in  25  and  ending  with  i  in 
1600.     The  last  solution  is  almost  colorless. 

Take  seven  test-tubes  of  thin,  colorless  glass,  and  as  uniform  as  possible 
in  diameter.  Number  them  i  to  7  and  two-thirds  fill  each  one  with  the 
dilute  blood  solution  corresponding  to  its  number.  Place  each  tube  before 
the  narrow  slit  of  the  spectroscope  and  adjust  the  flame  of  an  oil  or  gas 
lamp  so  that  its  light  may  pass  through  the  solution  into  the   slit.     Pull 


OPTICAL    PROPERTIES    OF    BLOOD. 


237 


out  the  draw  tube  until  the  light  is  properly  focused  and  observe  that  the 
bright  field  is  traversed  by  two  black  bands  which  cut  out  portions  of  the 


m  vo 


yellow  and  green.  With  strong  blood  solutions  all  light  except  red  is  shut 
out,  but  with  solutions  of  the  dilutions  2  to  7  the  field  is  obscured  only 
hy  the  two  bands.     In  solution  No.  2  they  are  very  dark  and  well  defined. 


238  PHYSIOLOGICAL    CHEMISTRY, 

With  increasing  dilution  they  grow  fainter  and  are  scarcely  visible  in  solu- 
tion No.  7.  In  all  the  solutions  examined  note  the  position  of  these  bands 
with  reference  to  the  characteristic  colors.  Note  also  that  the  bands  grow 
narrower  with  increasing  dilution,  and  that  it  becomes  more  and  more 
difficult  to  locate  the  edges  of  the  bands  sharply.  This  fact  has  some 
bearing  on  questions  of  quantitative  determinations  to  be  referred  to  later. 
Some  of  the  common  absorption  spectra  are  illustrated. 

With  instruments  furnished  with  a  simple  scale  it  soon  be- 
comes an  easy  matter  to  fix  approximately  the  limits  between 
which  each  band  is  found  and  also  the  point  of  deepest  absorp- 
tion in  each  band.  These  data  may  be  expressed  in  arbitrary 
scale  divisions,  in  fractions  of  the  distance  from  D  to  E,  or 
most  definitely,  in  wave  lengths  of  light,  if  the  instrument  has 
been  graduated  in  that  way.  In  all  accurate  comparisons  of 
spectra  some  such  method  of  recording  the  observations  must 
be  adopted. 

Spectrum  of  Reduced  Hemoglobin.  It  was  pointed  out 
in  the  last  chapter  that  the  spectrum  of  reduced  hemoglobin 
is  very  different  from  that  of  the  ordinary  oxyhemoglobin. 
In  place  of  two  bands  we  have  after  reduction  a  single  broad 
band  filling  three-fourths  of  the  space  between  D  and  E.  This 
is  the  simple  effect  observed  with  Stokes'  solution.  If  am- 
monium sulphide  is  employed  in  place  of  Stokes'  solution  the 
same  broad  band  appears  and  in  addition  a  single  narrow  band, 
the  center  of  which  is  in  the  red  to  the  left  of  D.  This  narrow 
band  may  be  due  to  some  sulphohemoglobin  formed  at  the 
same  time.  It  will  be  recalled  that  the  reduced  hemoglobin 
solution  is  purplish  red  in  place  of  deep  bright  red. 

Ex.  To  a  dilute  solution  of  blood,  about  i  part  to  50  of  water,  add  a 
few  drops  of  strong  ammonium  sulphide  solution  and  warm  gently  in  a 
test-tube  until  the  change  of  color  noted  above  is  reached.  Now  place  the 
tube  before  the  slit  of  the  spectroscope  and  observe  the  bands  referred 
to,  especially  the  narrow  one  in  the  red.  Hydrogen  sulphide  gives  prac- 
tically the  same  result. 

Ex.  Repeat  the  above  experiment,  using  Stokes'  solution  instead  of  the 
sulphide.  A  single  broad  band  appears  now ;  if  the  liquid  is  shaken  briskly 
the  air  acts  on  the  reduced  coloring  matter  with  oxidizing  effect,  as  shown 
by  a  division  of  the  band,  but  only  temporarily.  On  standing  a  short  time 
the  single  broad  band,  not  very  sharply  defined,  returns.     For  these  tests 


OPTICAL    PROPERTIES    OF    BLOOD.  239 

it  would  be  well  to  employ  several  dilutions,  beginning  with  No.  II  of  the 
series  given  aboA^e. 

Spectrum   of   Carbon   Monoxide   Hemoglobin.     It   has 

been  already  mentioned  that  the  spectrum  of  carbon  monoxide 
hemoglobin  is  very  similar  to  that  of  oxyhemoglobin.  This 
may  be  found  by  a  simple  test. 

Ex.  Into  diluted  blood,  as  before,  pass  a  stream  of  common  illuminat- 
ing gas  until  the  liquid  is  saturated,  which  requires  but  a  few  minutes. 
On  placing  the  tube  in  front  of  the  spectroscope  the  two  dark  bands  de- 
scribed will  be  seen,  and  slightly  farther  from  the  yellow  than  is  the  case 
with  oxyhemoglobin. 

These  bands  do  not  change  in  extent  or  position  by  agitation  of  the 
liquid  with  air,  as  follows  with  reduced  hemoglobin,  and  when  the  liquid 
is  treated  with  ammonium  sulphide  or  with  Stokes'  reagent  no  reduction 
takes  place.    The  two  bands  persist. 

Spectrum  of  Methemoglobin.  This  is  characterized  by  a  band  in  the 
yellowish  red  and  by  a  broad  band  in  the  blue.  To  some  moderately 
dilute  blood  add  a  few  drops  of  fresh  solution  of  potassium  ferricyanide. 
The  mixture  becomes  brown.  It  has  been  already  explained  that  by  care- 
ful reduction  with  a  small  amount  of  ammonium  sulphide  hemoglobin  is 
regenerated.  This  may  be  followed  by  the  spectroscope.  Add  a  few 
drops  of  the  sulphide  solution,  allow  the  mixture  to  stand  a  short  time 
and  then  shake  vigorously  in  the  air.     Oxyhemoglobin  bands  now  appear. 

The  Hematin  Spectra.  Of  these  the  spectrum  of  the  pigment  in  weak 
acid  mixture  is  the  most  characteristic.  This  may  be  obtained  by  first 
coagulating  10  cc.  of  blood  and  50  cc.  of  water  by  vigorous  boiling,  enough 
weak  sulphuric  acid  being  added  to  maintain  an  acid  reaction.  The  coag- 
ulum  is  separated,  pressed  dry  and  rubbed  up  in  a  mortar  with  25  cc.  of 
absolute  alcohol  and  i  cc.  of  strong  sulphuric  acid  gradually  added.  The 
mixture  is  then  transferred  to  a  flask  and  heated  half  an  hour  on  the 
water-bath.  After  cooling  the  filtered  liquid  may  be  examined.  A  strong 
dark  band  in  the  red  is  plainly  seen. 

Different  spectra  are  found  after  alkaline  treatment.  Warm  some  di- 
luted blood  with  a  few  drops  of  sodium  hydroxide  solution  until  a  brown- 
ish-green color  results.  The  absorption  spectrum  of  this  liquid  is  not 
characteristic.  The  whole  of  the  field  from  red  to  violet  is  dark.  On 
careful  reduction  with  a  little  ammonium  sulphide  or  Stokes'  solution  the 
spectrum  of  hemochromogen  or  reduced  hematin  may  be  obtained.  This 
consists  of  two  sharp  bands  between  D  and  E,  somewhat  like  those  of 
oxyhemoglobin,  but  nearer  E. 

QUANTITATIVE   SPECTRUM   ANALYSIS. 

The  amount  of  hemoglobin  in  a  solution  may  be  very  accurately  esti- 
mated  by   the   aid   of   the    spectroscope,   but   an   instrument    with    special 


HO 


PHYSIOLOGICAL    CHEMISTRY. 


attachments  for  the  purpose  is  required.  Several  distinct  methods  have 
been  applied  for  the  purpose,  but  the  methods  now  followed  involve  a 
simple  direct  comparison  between  light  which  has  been  weakened  by  pass- 
ing  through    a   blood    solution    and    light   passing    into   the    spectroscope 

directly.  Such  a  comparison  is 
easily  made  by  means  of  instru- 
ments with  double  collimator  slit 
as  first  introduced  by  Vierordt. 
The  arrangement  of  the  appa- 
ratus made  by  Krucss  is  shown 
in  Fig.  13,  while  the  double 
collimator  slit  and  ocular  and 
reading  scale  are  shown  in  Figs. 
16  and  17. 

The  method  of  measurement 
depends  on  the  principle  that 
there  is  a  simple  relation  be- 
tween the  amount  of  light  ab- 
sorbed by  a  solution  and  the 
concentration,  that  is  the  number 
of  absorbing  molecules  in  the 
same.  By  linding  therefore  the 
fraction  of  the  original  light 
absorbed  we  can  arrive  at  the  amount  of  absorbing  substance  in  solution. 
The  loss  of  light  in  passing  through  solutions  of  increasing  concentration 


Fig.    16.     Symmetrical    double    slit    for 
the  absorption  spectroscope. 


Fig.  17.     Ocular  and  reading  scale  of  the  Kruess  spectrophotometer. 

follows  the  law  worked  out  by  Lambert  for  the  loss  in  passing  a  series 
of  glass  plates  of  same  thickness  and  color.     Each  new  laver  absorbs  the 


OPTICAL    PROPERTIES    OF    BLOOD.  24 1 

same  fraction  of  the  light  reaching  it,  and  in  the  same  way  each  unit  of 
added  concentration  absorbs  the  same  fraction  absorbed  by  the  first  unit. 
Supposing  the  increased  absorption  of  light  to  follow  through  the  addi- 
tion of  new  layers  of  absorbing  substance,  the  relation  between  the  orig- 
inal and  residual  intensities  may  be  reached  in  this  manner.  Calling  the 
original  intensity  /  and  the  intensity  after  passing  the  first  layer  (or  first 
unit  of  concentration)  /'  we  have 

n 

the  original  intensity  being  reduced  to    —   by  the  first  layer.     By  a  second, 

third  and  following  layers  we  have 

II           III  I 

/ ,      / ,    /— . 

n      n  n      n      n  ?z'" 

The  last  expression  shows  the  intensity  after  passing  m  layers.     For  pur- 
poses of  calculation  this  can  be  put  in  another  form,  taking  the  original 

intensity  as  unity: 

I  log  / 

I'  =  ;;^i  gives  log  r  =  —  m  log  n.      log  ;z  =  —  —j^ ■ 

In  comparing  the  light-absorbing  powers  of  solutions  some  arbitrary 
basis  must  be  taken.  Practically  the  thickness  of  layer  which  will  reduce 
the  original  intensity  to  xV  i^s  value  is  so  taken.  The  light-extinguishing 
power  of  a  substance  or  its  coeMcient  of  extinction,  has  been  defined  as 
the  reciprocal  value  of  the  thickness  of  a  layer  of  the  substance  necessary  to 
reduce  the  intensity  of  the  transmitted  light  to  xV  *'^-^  original  value. 

Representing  the  extinction  coefficient  by  E  and  the  reduced  intensity  by 
/'  we  have  from  the  above  formulas : 


I  I 


jS  :^  —  and /'' = — '       log;? 
Therefore 


log  7:^ 
°  10 


10  °  I 


los/' 


In  practice  in  may  be  given  a  constant  value  and  called  i  (the  thickness 
of  cell,  for  example).    The  formula  becomes 

£  =  —  log  /'. 

It  was  said  above  that  increasing  the  thickness  of  a  layer  of  absorbing  sub- 
stance has  the  same  effect  as  increasing  its  concentration  in  the  same 
degree.  From  this  it  follows  that  the  extinction  coefficient  must  be  directly 
proportional  to  the  concentration.  Let  E  and  £'  represent  two  extinction 
coefficients  and  C  and  C  the  corresponding  concentrations,  then 

E   :  C  : :  £'  :  C. 
17 


342  PHYSIOLOGICAL    CHEMISTRY. 

The  relations 

E     E'     E'f 
c'   a'    C"' 

etc.,  must  be  all  equal  and  constant  for  the  same  substance.  This  con- 
stant ratio  is  a  characteristic  which  connects  the  light-absorbing  power 
of  a  solution  with  its  strength ;  it  may  be  represented  by  the  letter  A  and 
be  termed  the  absorption  ratio.     Hence,  for  a  given  color 

C 
^  =  E 

The  value  of  the  constant  A  must  be  found  for  a  given  spectrum  region 
by  employing  a  series  of  suitable  concentrations.  The  determination  of  E 
consists  in  finding  the  value  of  the  reduced  light  as  compared  with  the 
original ;  from  the  formula  given  above  the  extinction  coefficient  is  equal  to 
the  negative  logarithm  of  this  diminished  intensity.  In  the  various  forms 
of  photometers  employed  in  this  kind  of  work  the  peculiar  measuring 
mechanism  permits  the  direct  and  simple  estimation  of  the  intensity  of  the 
light  after  absorption  as  compared  with  the  light  before  absorption.  We 
find  /'  then  as  a  fraction,  and  E  is  the  negative  logarithm  of  this.  For 
example,  suppose  we  have  in  a  liter  0.25  gm.  of  an  absorbing  substance. 
The  concentration,  or  C,  is  0.00025.  which  represents  the  value  per  cubic 
centimeter,  taken  as  the  unit  of  volume.  Next,  suppose  we  find  with  our 
special  measuring  instrument  that  the  value  of  the  light  after  absorption  is 
only  0.0436  of  the  original,  that  is  about  5'.^.  Substituting  in  our  formula 
we  have 

£  =  —  log  /'  =  —  log  0.0436  =  1. 3605 1 
and  finally 

C      0.00025 

^=^=t:36^=°-°°°^^4. 

In  this  way,  by  repeating  the  observations  with  a  number  of  different 
strengths  of  solution  of  the  substance,  we  find  the  value  of  the  constant. 
As  the  individual  observations  may  differ  a  little  the  mean  must  be  taken. 
A  becomes  thus  fixed  once  for  all  for  a  given  spectral  region,  and  its  value 
may  be  employed  to  determine  the  concentration  of  imkno'ci'ii  solutions  since 

C  =  EA. 
Quantitative  spectrum  analysis  by  absorption  is  based  on  these  very 
simple  principles.  Blood  or  other  .substance  to  be  examined  is  placed  in  a 
cell  with  plane  parallel  sides,  preferably  exactly  i  cm.  apart.  The  cell 
should  be  half  filled  and  is  brought  into  proper  position  in  front  of  a 
spectroscope  with  a  double  slit,  the  level  of  the  liquid  just  reaching  to  the 
top  of  the  lower  slit.  The  light  from  the  illuminating  lamp  enters  the 
upper  slit  directly  and  the  lower  one  through  the  colored  liquid.  If  the 
two  slit  openings  were  the  same  to  begin  with  the  upper  one  must  now  be 
narrowed  until  the  light  passing  through  it  is  reduced  to  the  intensity  of 
the  light  through  the  blood  and  the  lower  slit.  The  width  of  each  slit 
may  be  measured  on  a  micrometer  screw  head  or  in  some  other  convenient 


OPTICAL    PROPERTIES    OF    BLOOD. 


243 


way.  In  these  observations  but  a  small  portion  of  the  spectrum  is  brought 
into  the  field  of  view.  The  eyepiece  in  the  spectroscope  is  therefore  fur- 
nished with  a  screen  which  can  be  opened  or  narrowed  at  will  and  sym- 
metrically, that  is  from  both  sides,  so  as  to  expose  some  definite  small  por- 
tion of  the  spectrum.  The  instrument  should  be  so  constructed  as  to 
permit  any  desired  portion  of  the  spectrum  to  be  quickly  and  accurately 
brought  into  the  field. 

In  place  of  using  a  simple  cell  it  is  much  better  in  practice  to  employ  a 
cell  with  so-called  Schulz  glass  prism.     With  the  simple  cell  the  meniscus 


Cb 

\ 

J 

Fig.  18.  Fig.  19. 

Absorption   cell   and    Schulz    glass   prism   as   used   in   quantitative   analysis   by 
absorption.     The  position  of  the  prism  is  shown  in  Fig.   19. 

formed  at  the  top  of  the  liquid  projects  a  broad  dark  band  across  the  field 
horizontally  which  makes  the  comparison  of  the  upper  and  lower  spectra 
very  difficult.  With  the  cell  furnished  with  a  Schulz  prism  this  difficulty 
is  largely  overcome,  but  the  details  of  the  arrangement  can  not  be  explained 
here.  They  will  be  easily  understood  by  use  of  the  instrument.  When  the 
Schulz  prism  is  employed  light  enters  the  upper  slit  through  i.i  cm.  of 
solution  and  the  lower  slit  through  o.i  cm.  of  solution  and  the  i.o  cm.  of 
the  clear  glass  prism. 


Name  of  Substance. 

Spectral  Region 

A  = 

Oxyhemo- 

Hemo- 

Methemo- 

Co-Hemo- 

Bilirubin in 

Bilirubin  in 

g^lobin. 

globin. 

globin. 

globin. 

Chloroform. 

Alcohol. 

569-3-555.5 

0.00133 

0.00122 

0.00260 

O.OOI3I 

549.9-540.0 

0.00 1 00 

0.00150 

0.00199 

O.OOII5 

558.1-534.3 

O.OOII3 

0.000215 

501.2-494.3 

0.0000598 

0.000142 

494.3-486.1 

0.0000356 

0. 0001 16 

486.1-480.6 

0.0000209 

0.000102 

480.6-474.4 

0.0000148 

0.0000842 

474.4-468.4 

0.0000126 

0.0000700 

468.4-461.7 

0.0000 II 8 

0.0000667 

244 


PHYSIOLOGICAL    CHEMISTRY 


The  absorption  ratios  for  a  number  of  physiologically  important  sub- 
stances are  shown  in  the  above  table.  The  spectral  regions  are  given 
in  the  usual  wave  lengths. 


CLINICAL  METHODS  OF  ESTIMATING  OXYHEMOGLOBIN. 

The  spectrophotometric  estimation  of  oxyhemoglobin  as 
described  above  is  not  simple  enough  for  quick  clinical  deter- 
minations, which  have  to  be  made  in  the  course  of  daily  prac- 
tice by  medical  men.  Other  forms  of  apparatus  ha\'e  been 
devised  for  this  purpose  and  are  in  common  use.  In  all  of 
these,  comparison  is  made  between  the  blood  under  examina- 
tion, properly  diluted,  and  a  standard  color  assumed  to  repre- 
sent normal  blood  correspondingly  diluted.  Some  of  these 
appliances  give  pretty  good  results,  but  others  are  very  faulty 
and  the  values  they  furnish  quite  untrustworthy.  In  the  fol- 
lowing pages  several  of  the  commoner  forms  will  be  briefly 
described. 

Fleischl's  Hemometer.  This  instrument  consists  essentially  of  a  circular 
cell  with  glass  bottom  divided  by  a  vertical  partition  into  two  equal  com- 
partments as  shown  below.     In  one  of  these  the  accurately  diluted  blood 

is  placed  in  given  volume.  The 
other  compartment  is  filled  with  pure 
water  to  the  same  level.  The  cell 
rests  on  a  stage  below  which  there 
is  mounted  a  w'hite  reflecting  mirror 
by  means  of  which  light  may  be 
thrown  upward  to  illuminate  the  two 
compartments  of  the  cell  uniformly. 
Immediately  under  the  water  com- 
partment a  long  colored  glass  wedge 
is  placed  in  such  a  manner  that  light 
must  pass  through  it  into  the  water. 
By  a  rack  and  pinion  mechanism  this 
wedge  may  be  moved  to  the  right  or 
left  under  the  w'ater  cell  so  as  to 
bring  a  thinner  or  thicker  portion 
of  the  glass  below  the  water.  The 
glass  is  colored  by  means  of  purple 
of  Cassius  to  resemble  diluted  blood 
as  nearly  as  possible,  and  the  light  shining  through  it  into  the  water  im- 
parts  a   more   or  less  perfect  blood   color   to   it.     The   wedge    is   moved 


Fig.  20.  Fleischl  hemometer, 
showing  divided  cell  for  blood  and 
water  and  reflecting  mirror  to 
secure  uniform    illumination. 


OPTICAL    PROPERTIES    OF    BLOOD. 


245 


until  the  liquids  in  the  two  compartments  of  the  cell  appear  to  have  the 
same  color.  The  color  in  a  certain  portion  of  the  wedge  is  intended  to 
correspond  to  normal  blood,  or  blood  with  100  per  cent  of  the  normal 
oxyhemoglobin,  and  degrees  placed  at  proper  intervals  along  the  wedge 
represent  corresponding  higher  or  lower  percentages.  For  example,  if 
when  the  colors  in  the  two  compartments  are  matched  the  reading  on  the 
wedge  scale  is  97,  it  means  that  the  blood  contains  color  due  to  97  per  cent 
of  the  normal  or  average  oxyhemoglobin  content. 


/^ 


Fig.  21.  Dare's  hemoglobinometer.  R, 
milled  wheel  acting  by  a  friction  bearing  on 
the  rim  of  the  color  disc ;  S,  case  inclosing 
color  disc,  and  provided  with  a  stage  to  which 
the  blood  chamber  is  fitted  ;  T,  movable  wing 
which  is  swung  outward  during  the  observa- 
tion, to  serve  as  a  screen  for  the  observer's 
eyes,  and  which  acts  as  a  cover  to  inclose  the 
color  disc  when  the  instrument  is  not  in  use ; 
U,  telescoping  camera  tube,  in  position  for 
examination ;  V,  aperture  admitting  light  for 
illumination  of  the  color  disc ;  X,  capillary 
blood  chamber  adjusted  to  stage  of  instru- 
ment, the  slip  of  opaque  glass,  W,  being 
nearest  to  the  source  of  light ;  Y ,  detachable 
candle-holder ;  Z,  rectangular  slot  through 
which  the  hemoglobin  scale  indicated  on  the 
rim  of  the  color  disc  is  read. 


Fig.  22.  Horizontal  Sec- 
tion of  Dare's  Hemoglobin- 
ometer, in  which  the  arrange- 
ment of  parts  is  clearly 
shown.  L  is  the  standard 
wedge-shaped  color  disc. 
The  blood  is  inclosed  between 
the  glass  plates  0  and  P  and 
is  illuminated  by  the  flame  /. 
The  eye  observes  the  blood 
and  the  color  scale  through 
the  apertures  M  and  M'. 


This  Fleischl  instrument  is  one  of  the  best  in  principle  but  it  must  be 
carefully  used  to  furnish  good  results.  The  color  of  the  wedge  does  not 
correspond  to  blood  color  unless  a  certain  kind  of  white  light  is  employed. 


246  PHYSIOLOGICAL    CHEMISTRY. 

A  wedge  made  for  candle  light  cannot  be  used  with  sunlight.  In  addition 
to  this  difficulty  the  wedges  themselves  are  often  at  fault.  They  sometimes 
fail  to  produce  a  blood  red  with  any  kind  of  light. 

Miescher  has  suggested  several  improvements  in  the  Fleischl  instrument 
which  render  it  much  more  accurate.  Readings  may  be  made  from  several 
different  dilutions   from   which   a  mean  value  may  be   taken. 

The  Hemoglobinometer  of  Gowers.  This  instrument  has  been  made  in 
several  forms.  The  construction  is  essentially  this.  Two  narrow  glass 
tubes  of  the  same  diameter  are  used ;  one  receives  as  a  standard  a  i  per 
cent  solution  of  normal  blood,  while  the  other  is  graduated  from  below 
from  o  to  100  degrees  and  is  intended  to  receive  the  blood  under  examina- 
tion. A  measured  portion  of  this  blood,  usually  20  cubic  millimeters,  is 
poured  into  the  tube  and  diluted  with  distilled  water,  a  little  at  a  time. 
After  each  addition  of  water  the  tube  is  shaken  thoroughly  to  mix  and  a 
comparison  made  with  the  standard  tube.  When  the  colors  are  finally  the 
same,  as  read  horizontally,  that  is  across,  not  down  through  the  tubes,  the 
degree  of  dilution  reached  in  the  graduated  tube  is  noted.  This  indicates 
the  percentage  of  color  present  as  compared  with  the  standard.  A  blood 
which  can  be  diluted  to  100  degrees  (100  times  the  original  small  volume 
taken)  contains  100  per  cent  of  the  normal  hemoglobin  content  or  is  nor- 
mal, while  if  it  can  be  diluted  to  75  degrees  only,  the  comparison  shows 
that  this  blood  contains  but  75  per  cent  of  the  average  hemoglobin. 

In  place  of  using  blood  as  a  standard  a  gelatin  solution  stained  with  picro- 
carmine  or  other  stain  is  frequently  employed.  But  in  time  such  color 
standards  always  fade,  and  an  abnormally  high  result  is  recorded  as  a 
consequence. 

Dare's  Hemoglobinometer.  In  this  instrument  the  principle  employed  in 
the  Fleischl  apparatus  is  used,  but  the  comparison  is  made  between  the 
colored  glass  standard  and  undiluted  blood.  The  possible  error  due  to 
dilution  is  thus  avoided.  Some  idea  of  the  apparatus  is  given  by  the 
illustrations  above.  A  drop  of  perfectly  fresh  blood  is  placed  over  the 
opening  in  a  capillary  flat  cell  into  which  it  is  immediately  drawn,  much 
as  a  drop  of  water  is  drawi  in  between  a  slide  and  cover  glass  not  in 
absolute  close  contact,  when  the  water  is  put  on  the  edge  of  the  cover. 
The  capillary  observation  cell  is  mounted  at  the  end  of  an  eyepiece  through 
which  it  may  be  clearly  seen.  A  small  portion  of  the  colored  glass  standard 
may  be  seen  at  the  same  time.  The  blood  cell  and  red  glass  are  evenly 
illuminated  by  a  candle  flame  placed  in  fixed  position  in  front  of  the  appa- 
ratus. The  colored  glass  standard  is  given  the  form  of  a  circular  disk, 
which  may  be  rotated  by  a  screw  motion.  This  disk  is  beveled  from  one 
side  to  the  other,  giving  a  wedge  effect  as  in  the  Fleischl  apparatus.  The 
rotation  of  the  disk  brings  therefore  thicker  or  thinner  portions  of  the  edge 
in  the  field  of  the  eyepiece  along  with  the  cell  holding  the  blood.  When 
the  colors  are  matched  the  corresponding  hemoglobin  value  is  read  off  on 
a  scale.  In  practice  this  instrument  is  somewhat  more  convenient  than  the 
Fleischl.    The  accuracy  is  about  the  same. 


OPTICAL    PROPERTIES    OF    BLOOD.  247 

Tallquist's  Chart.  This  consists  of  a  small  book  containing  blank  sheets 
of  a  special  fine  grained  filter  paper  and  a  colored  chart  with  a  number  of 
shades  corresponding  to  blood  stains  with  different  hemoglobin  content. 
To  make  the  test  a  small  drop  of  blood  is  drawn  and  placed  on  one  of 
the  sheets  of  filter  paper,  into  which  it  soaks  and  spreads.  The  color  pro- 
duced is  compared  with  one  of  the  ten  shades  in  the  color  scale. 

The  test  is  extremely  simple,  but  its  accuracy  is  dependent  on  the 
accuracy  with  which  the  colors  in  the  chart  are  printed,  and  their  perma- 
nence. Unfortunately  the  colors  change  as  the  chart,  in  use,  is  exposed  to 
light  and  air. 


CHAPTER    XIII. 

FURTHER   PHYSICAL   METHODS    IN    BLOOD    EXAMINATION. 

FREEZING   POINT   AND  ELECTRICAL  CONDUCTIVITY. 

THE  HEMATOCRIT. 

OSMOTIC  PRESSURE. 

In  many  of  the  phenomena  of  the  body  the  osmotic  pressure 
of  dissolved  substances  plays  an  extremely  important  part. 
This  is  especially  true  in  the  study  of  the  blood  as  a  whole, 
and  it  is  therefore  proper  at  this  point  to  enter  upon  a  short 
explanation  of  what  is  meant  by  osmotic  pressure  and  what 
its  relations  are. 

Nature  of  Osmotic  Pressure.  Solids  in  solution  exert  a 
pressure  in  all  directions  quite  analogous  to  that  observed  with 
gases,  and  in  general  the  laws  connecting  increase  in  pressure 
with  concentration  and  temperature  are  the  same  as  for  gases. 
With  many  solids,  however,  dissociation  in  solution  or  separa- 
tion into  ions  takes  place  and  each  separate  ion  behaves  as  a 
whole  molecule  as  far  as  pressure  is  concerned. 

Some  of  the  simple  effects  of  this  pressure  are  easily  ob- 
serv-ed.  When  a  drop  of  a  strong  solution  of  blue  vitriol  is 
placed  carefully  on  the  surface  of  a  weak  solution  of  potassium 
ferrocyanide  a  precipitate  of  copper  ferrocyanide  forms  as  a 
sheath  or  membrane  around  the  vitriol  drop  and  holds  it  in 
nearly  spherical  form.  If  the  drop  is  properly  deposited, 
which  requires  some  care,  it  will  gradually  enlarge  by  the  en- 
trance of  water,  which  dilutes  the  enclosed  copper  sulphate. 
None  of  the  latter  passes  out  and  the  ferrocyanide  solution 
evidently  does  not  enter  since  no  more  precipitate  forms  within 
the  drop.  The  copper  ferrocyanide  membrane  must  possess 
therefore  some  interesting  properties ;  it  is  permeable  for 
water,  it  is  not  permeable  for  either  of  the  salt  solutions.  Sim- 
ilar membranes  may  be  made  with  a  number  of  substances  and 

248 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION. 


249 


their  impermeability  for  many  salt  or  other  solid  molecules 
may  be  shown.  Some  membranes  are  permeable  for  certain 
salts  but  not  for  others.  The  fact  of  the  existence  of  pressure 
within  such  a  membrane  may  be  shown  by  the  following  well- 
known  experiment,  in  which  a  copper  ferrocyanide  sheath  or 
membrane  is  made  in  a  different  manner. 

Ex.  Procure  a  small  fine  grained  porous  battery  cell,  about  3  to  4  inches 
long  and  i  inch  in  outside  diameter,  and  clean  it  thoroughly.  This  may  be 
done  by  washing  the  cell  with  water,  then  with  weak  hydrochloric  acid  and 
finally  with  water  very  thoroughly.  Close  the 
cell  with  a  perforated  rubber  stopper,  pass  a 
glass  tube  through  the  perforation  and  con- 
nect the  outer  end  of  this  with  a  suction  pump. 
On  dipping  the  cell  in  water  or  the  acid  it 
may  be  drawn  through  the  pores  of  the  cell 
to  effect  the  cleaning.  When  the  cell  is  clean 
it  is  placed  in  a  potassium  ferrocyanide  solu- 
tion containing  about  150  gm.  per  liter  and 
solution  drawn  through  by  means  of  the  pump 
until  the  pores  are  thoroughly  filled.  Then 
the  cell  is  washed,  inside  and  out,  with  dis- 
tilled water  and  immersed  in  a  blue  vitriol 
solution  containing  about  250  gm.  per  liter.  A 
precipitate  is  thus  formed  within  the  pores  of 
the  cell,  which  is  allowed  to  remain  some 
hours  in  the  solution.  The  cell  is  then  re- 
moved, washed  with  water  and  is  ready  for 
use.  Fill  it  with  a  5  per  cent  cane  sugar  solu- 
tion, close  with  the  rubber  stopper  and  long 
narrow  glass  tube  and  immerse  the  cell  in  a 
beaker  of  distilled  water  the  'temperature  of 
which  should  be  the  same  as  that  of  the  sugar 
solution.  After  a  short  time  liquid  begins  to 
rise    in    the    glass    tube    which    serves    as    a 

kind  of  manometer.  This  is  in  consequence  of  the  entrance  of  water  to 
the  sugar  solution.  Sugar  can  not  pass  out  in  the  other  direction  as  the 
precipitate  membrane  is  not  permeable  for  it,  but  it  is  readily  permeable 
for  the  water.  The  sugar  in  its  effort  to  pass  out  to  the  water  exerts  a 
pressure  on  the  retaining  membrane,  and  it  is  because  of  this  pressure 
that  the  water  is  able  to  enter  the  cell.  The  flow  of  the  water  continues 
until  its  hydrostatic  pressure  exactly  balances  the  sugar  or  osmotic  pres- 
sure. In  some  cases  mercury  manometers  attached  to  such  cells  register 
pressure  of  several  atmospheres. 

The  pressure  actually  observed  in  such  an  apparatus  is  just  short  of 
that  required  to  press  the   solvent,  water,  through   the  membrane  in  the 


Fig.  23.  Apparatus  for 
observing  and  measuring 
osmotic   pressure. 


250  PHYSIOLOGICAL    CHEMISTRY. 

opposite  direction.  Theoretically  it  should  amount  to  22.4  atmospheres 
for  a  solution  containing  a  gram  molecular  weight  dissolved  per  liter, 
since  it  has  been  found  that  the  osmotic  pressure  of  a  body  is  the  same 
it  would  possess  if  it  existed  in  the  condition  of  a  gas  at  the  same  tem- 
perature and  in  the  same  volume.  A  gram  molecular  weight  of  hydro- 
gen (2.014  gnis.),  of  oxygen  (32  gms.),  of  nitrogen  or  other  gas  occupies 
a  volume  of  22.4  liters  under  normal  temperature  and  pressure  condi- 
tions. If  condensed  into  i  liter  their  pressure  would  be  22.4  atmospheres. 
Experiment  has  shown  that  a  gram  molecular  weight  of  sugar  or  similar 
solid  in  water  to  make  a  liter  volume  e.xerts  a  pressure  of  22.4  atmos- 
pheres. In  the  case  of  salts  which  break  up  into  component  parts  or 
ions  the  pressure  becomes  correspondingly  greater.  In  very  dilute  solu- 
tions a  molecule  of  sodium  chloride,  for  example,  exerts  practically 
double  the  pressure  observed  for  a  molecule  of  sugar.  In  this  dilute  con- 
dition the  component  parts,  or  ions,  of  sodium  and  chlorine  seem  to  exert 
a  pressure  corresponding  to  whole  molecules. 

The  above  experiment  is  a  somewhat  crude  one  and  is  in- 
tended merely  as  an  illustration  of  the  development  of  pres- 
sure. For  accurate  measurements  much  more  elaborate  ap- 
paratus must  be  employed  and  numerous  precautions  observed. 
Practically,  however,  osmotic  pressure  is  always  measured  by 
indirect  methods  to  be  explained  later.  A  familiar  illustration 
of  a  semi-permeable  sheath  or  membrane  is  found  in  the  red 
blood  corpuscle.  Normally  this  holds  its  hemoglobin  and  cer- 
tain salts  because  it  is  suspended  in  a  liquid  which  has  the 
same  osmotic  pressure.  But  if  the  corpuscles  be  placed  in 
pure  water  they  are  seen  to  swell  and  finally  break  because  of 
the  passage  of  water  through  the  cell  sheath  which  is  not 
permeable  for  the  solid  contents.  By  means  of  the  hematocrit, 
as  Avill  be  explained,  it  is  possible  to  find  the  average  volume 
occupied  by  the  corpuscles  in  a  given  sample  of  blood.  When 
mixed  with  water  or  solutions  with  lower  osmotic  pressure 
the  corpuscle  volume  increases ;  in  stronger  salt  solutions,  on 
the  other  hand,  the  individual  corpuscles  shrink  in  size  and 
their  total  volume  becomes  less.  The  hematocrit  may  there- 
fore be  used  to  measure  or  compare  osmotic  pressures  in  cer- 
tain cases. 

INDIRECT   METHODS.     CRYOSCOPY. 

Although  the  blood  contains  about  20  per  cent  of  organic 
substances  and  about  i  per  cent  of  mineral  matters  its  osmotic 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION.  25  I 

pressure  depends  largely  on  the  latter.  This  is  because  of  the 
simple  fact  that  the  large  gross  weight  of  organic  matter  rep- 
resents relatively  but  a  small  number  of  molecules,  and  the 
actual  pressure  is  measured  by  the  total  number  of  molecules 
or  ions  present.  This  osmotic  pressure  in  health  remains 
practically  constant;  even  after  great  loss  of  blood  when  the 
total  volume  is  restored  by  drawing  on  the  lymph  serum,  al- 
though the  relative  number  of  corpuscles  may  be  much  re- 
duced, the  osmotic  pressure  of  the  new  blood  is  practically 
unchanged.  This  is  due  to  the  fact  that  the  blood  and  lymph 
serum  are  isosmotic. 

The  Freezing  Point  Method,  The  measurement  of  the 
osmotic  pressure  of  blood  or  any  other  solution  by  the  direct 
method  suggested  by  the  experiment  given  above  is  extremely 
difficult.  Several  indirect  methods  may  be  followed,  two  of 
which  are  in  common  application.  One  of  these  only  is  suit- 
able for  the  examination  of  blood.  In  the  first  of  these  meth- 
ods the  elevation  of  the  boiling  point  of  the  solution  is  ob- 
served; in  the  second  the  depression  of  its  freezing  point. 
Comparatively  simple  relations  obtain  between  the  three  phe- 
nomena. In  a  solution  the  tension  of  the  vapor  is  decreased 
in  proportion  as  the  osmotic  pressure  of  the  dissolved  sub- 
stance increases  and  more  heat  must  therefore  be  applied  to 
actually  lift  the  atmosphere  or  boil  off  the  solvent.  For  each 
gram-molecule  per  liter  dissolved  this  elevation  of  the  boiling 
point  of  water  is  about  0.52°.  This  method,  by  noting  the 
elevation  of  the  boiling  point,  cannot  be  applied  to  blood,  be- 
cause of  its  coagulation,  but  there  is  no  drawback  to  the 
method  depending  on  the  separation  of  the  solvent  by  freez- 
ing. With  increase  in  amount  of  salt  or  foreign  substance 
dissolved  in  the  water,  the  lower  must  its  temperature  be 
brought  to  effect  a  partial  separation  by  freezing  out  a  portion 
of  the  solvent.  The  lowering  of  the  freezing  point  is  accu- 
rately proportional  to  the  number  of  molecules  (or  ions)  pres- 
ent. The  molecular  freezing  point  depression  for  water  is 
1.85'';  that  is  the  freezing  point  of  a  solution  containing  one 


2  c  2 


PHYSIOLOGICAL    CHEMISTRY. 


'9 


Fig.  24.  Beckmann  freez- 
ing point  apparatus.  D  is 
a  fine  thermometer,  C  the 
containing  jar,  B  the  out- 
side or  air  mantle  tube  and 
A  the  tube  in  which  the 
mixture  to  be  observed  is 
placed.  Two  stirrers  are 
shown  ;  one  for  the  cooling 
mixture  in  the  jar  and  one 
for  the  experimental  mix- 
ture. 


molecular  weight  in  grams  of  a  sub- 
stance, such  as  sugar  or  urea,  dis- 
solved in  a  liter,  is  1.85°  below  the 
freezing-point  of  water.  The  os- 
motic pressure  of  a  substance  of 
which  I  gram  molecule  per  liter  is 
dissolved  in  water,  is  22.4  atmos- 
pheres. Therefore  a  freezing  point 
depression  of  i^  C.  corresponds  to 
an  osmotic  pressure  of  12.1  atmos- 
pheres. 

Apparatus.  Various  forms  of  apparatus 
have  been  devised  for  the  experimental  de- 
termination of  freezing  point.  The  Beck- 
mann apparatus  is  most  commonly  employed. 
It  consists  essentially  of  a  strong  test-tube 
to  contain  the  substance  to  be  examined. 
This  is  suspended  in  a  somewhat  larger  tube 
which  serves  as  an  air  bath.  The  large  tube 
finally  is  supported  in  a  strong  beaker  or 
battery  jar  which  receives  the  freezing  mix- 
ture to  reduce  the  temperature  of  the  sub- 
stance under  experiment.  The  freezing 
mixture  may  consist  of  ice,  water  and  salt, 
which  must  be  stirred  up  frequently  to  main- 
tain a  uniform  degree,  of  cold.  A  very  deli- 
cate thermometer  passes  down  into  the  sub- 
stance in  the  inner  tube,  which  is  also  fur- 
nished with  a  stirrer  of  platinum  wire.  The 
blood  or  other  liquid  is  stirred  until  coagu- 
lation begins,  the  thermometer  being  mean- 
while carefully  watched.  The  temperature 
goes  down  at  first  a  little  below  the  normal 
freezing  point,  because  of  overcooling,  but 
soon  rises  and  remains  stationar\-.  In  ex- 
perimenting with  aqueous  solutions  a  known 
weight  of  pure  water  is  taken  and  its  freez- 
ing point  with  the  thermometer  used  is  accu- 
rately found.  Then  the  salt  or  other  body, 
which  has  been  accurately  weighed,  is  added 
and  a  new  determination  made.  While  the 
principle  is  simple  the  details  call  for  some 
skill  in  manipulation.  Full  descriptions  of 
the    method    may    be    found    in    works    oi>; 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION.  253 

physical  or  organic  chemistry,  as  it  finds  applications  in  many  directions, 
and  especially  in  the  determination  of  molecular  weight.  The  general 
appearance  of  the  simple  apparatus  is  here  shown. 

The    freezing    point    of    normal    human    blood    is    about 

—  0.56°.  As  a  reduction  of  i°  corresponds  to  an  atmospheric 
pressure  of  12.  i  atmospheres,  the  normal  osmotic  pressure  of 
the  blood  is  about  6.8  atmospheres.  It  makes  but  little  differ- 
ence here  whether  we  consider  the  whole  blood  or  the  plasma 
free  from  corpuscles  and  fibrin.  The  result  is  mainly  due  to 
the  small  molecules  present,  and  these  are  inorganic.  A  solu- 
tion of  20  gms.  of  serum  albumin  in  water  to  make  100  cc. 
would  have  a  freezing  point  of  about  — 0.03°;  the  effect  of 
the  other  proteins  would  be  practically  the  same.  A  solution 
of  urea  containing  10  gm.  in  100  cc.  has  a  freezing  point  of 
■ — 3-o8°.  one  of  glucose  with  10  gm.  in  100  cc.  a  freezing 
point  of  —  1 .03°,  while  a  solution  of  common  salt  with  the 
same  weight  dissolved  would  show  a  depression  of  about  5°. 

Variations.  This  observed  freezing  point  depression  is 
normally  constant  and  nearly  the  same  for  the  blood  of  all  the 
common  animals.  But  temporary  variations  may  occur. 
After  consumption  of  large  quantities  of  water  it  may  sink  to 

—  0.51°,  while  following  a  meal  rich  in  salty  food  a  further 
depression  to  — 0.62°,  or  even  lower,  may  be  observed.  But 
these  changes  are  very  speedily  rectified  through  the  elimina- 
tion of  proper  quantities  of  salts  and  water  by  the  kidneys. 
If  an  examination  of  the  blood  shows  a  greater  depression  than 
that  which  may  be  accounted  for  by  absorption  of  food  con- 
stituents a  failure  of  some  kind  in  the  functions  of  the  kidneys 
is  indicated.  Through  injury  to  the  mechanism  of  these  or- 
gans the  osmotic  pressure  of  the  blood  may  rise  to  over  12 
atmospheres,  corresponding  to  a  depression  of  the  freezing 
point  of  a  whole  degree  or  more. 

Because  of  these  observed  facts  the  determination  of  the 
freezing  point  of  the  blood  has  become  a  test  of  practical  im-- 
portance  in  the  diagnosis  of  disorders  of  the  kidney.  With 
proper  facilities  the  experiment  may  be  quickly  made  and  will 


254  PHYSIOLOGICAL    CHEMISTRY. 

sen^e  to  detect  an  abnormality  in  the  blood  more  readily  than 
this  may  be  accomplished  by  chemical  analysis.  It  is  custo- 
mary at  the  present  time  to  designate  this  freezing  point  de- 
pression by  A.     Thus,  normally,  for  human  blood 

A  3= -0.56°. 

ISOTONIC   COEFFICIENT. 

When  a  few  drops  of  blood  are  mixed  with  an  excess  of 
salt  solution  of  a  certain  strength  and  the  mixture  allowed  to 
stand  at  rest  the  corpuscles  gradually  settle  and  leave  a  color- 
less liquid  above.  If  the  same  volume  of  a  certain  weaker 
salt  solution  is  taken  with  the  blood  the  mixture  after  shaking 
is  found  to  leave  no  longer  a  colorless  liquid  above  the  settled 
corpuscles,  but  a  somewhat  reddish  liquid.  This  color  shows 
that  the  corpuscles  have  been  broken  and  that  a  little  of  the 
hemoglobin  has  escaped.  An  experiment  will  illustrate  the 
fact:  it  is  due  to  Hamburger. 

Ex.  Prepare  a  series  of  common  salt  solutions  of  the  following 
strengths :  0.7  per  cent,  0.65  per  cent,  0.60  per  cent,  0.55  per  cent,  0.50  per 
cent  and  0.45  per  cent.  Measure  out  accurately  20  cc.  of  each  into  test- 
tubes  and  add  to  each  5  drops  of  defibrinated  bullock's  blood.  Shake  and 
allow  to  stand.  Notice  that  in  some  of  the  tubes  the  corpuscles  have 
settled,  leaving  the  salt  solution  practically  clear  and  colorless ;  in  others 
there  is  color,  which  is  greatest  in  the  tube  with  the  least  salt.  In  the 
tube  with  0.60  per  cent  of  salt  there  should  be  no  color,  while  in  the  tube 
with  the  next  weaker  solution  some  appears.  There  must  be  therefore 
some  solution  between  these  two  in  which  the  corpuscles  just  fail  to  give 
up  color.     Hamburger  found  this  to  be  one  with  0.58  per  cent  of  salt. 

Osmotic  Tension.  Hamburger  made  a  large  number  of 
experiments  of  this  description  and  found  the  limiting  value 
of  the  strength  of  solutions  for  which  no  loss  of  color  follows. 
He  spoke  of  these  solutions  as  being  isotonic,  or  as  having  the 
same  osmotic  tension  as  the  contents  of  the  corpuscles.  The 
numerical  values  found  bear  a  close  relation  to  the  molecular 
weights  of  the  salts  used.  Thus,  the  following  values  were 
noted : 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION.  255 

Molecular  Weight  Isotonic  Value 

NaCl    58.5  0.58s  per  cent. 

NaBr    103.0  1.02 

Nal    149.9  i-SS 

KNO3    101.2  I.OI 

KBr    119.1  1. 17 

KI  166.0  1.64 

These  values  while  isotonic  and  isosmotic  with  each  other 
are  not,  however,  quite  isosmotic  with  the  blood.  A  common 
salt  solution  having  a  percentage  strength  of  0.9  per  cent 
has  practically  the  same  freezing  point  as  the  blood.  Blood 
corpuscles  (human)  in  such  a  solution  do  not  swell  or  shrink, 
consequently  lose  no  hemoglobin.  But  if  placed  in  weaker 
salt  solutions  water  is  gradually  absorbed  to  make  the  outside 
and  inside  osmotic  pressure  the  same.  x\fter  a  time,  however, 
with  decreasing  strength  of  the  salt  solution,  so  much  water 
is  absorbed  that  the  limit  of  strength  of  the  corpuscle  sheath 
is  reached  and  a  break  follows.  The  escape  of  hemoglobin 
shows  this  point.  With  salt  solutions  this  break  takes  place 
with  practically  corresponding  osmotic  pressures,  but  there  are 
many  substances  which  do  not  follow  the  rule  at  all.  This  is 
particularly  true  of  solutions  of  urea,  glycerol,  ammonium 
carbonate,  sodium  carbonate  and  ammonium  chloride.  Even 
with  rather  strong  solutions  of  these  bodies  the  corpuscles  fail 
to  hold  their  hemoglobin.  A  satisfactory  explanation  of  the 
abnormal  behavior  of  these  bodies  is  not  known.  Blood  so 
changed  is  said  to  be  lake-colored.  Following  the  Hamburger 
designations  normal  blood  was  said  to  be  hyperisotonic,  since 
it  contains  more  than  enough  salts  to  hold  the  corpuscle  intact. 

HEMATOCRIT  METHODS. 

It  has  been  shown  above  that  the  red  blood  corpuscles  main- 
tain their  normal  volume  in  liquids  which  have  the  same  os- 
motic pressure  as  the  blood.  In  liquids  with  a  lower  pressure 
they  swell,  while  in  solutions  possessing  a  higher  osmotic 
pressure  than  the  blood  they  contract.  The  corpuscles  are 
extremely  sensitive  to  such  influences,  and  changes  in  volume 


256  PHYSIOLOGICAL    CHEMISTRY. 

folloAV  with  even  very  trifling  changes  in  the  osmotic  pressure 
of  a  Hquid  with  which  the  blood  may  be  mixed.  The  blood 
corpuscle  may  be  used  then  as  a  kind  of  indicator  to  disclose 
variations  in  osmotic  pressure,  and  substances  may  be  com- 
pared as  to  the  osmotic  pressure  they  exert  bv  noting  their 
behavior  with  the  corpuscles. 

It  Avould  of  course  be  very  difficult  to  prove  anything  by 
measurements  on  a  single  corpuscle,  but  it  is  possible  to  make 
the  observation  on  a  large  \-olume.  If  blood  is  drawn  up  into 
a  narrow  tube  of  capillary  dimensions,  placed  in  a  centrifuge 
and  rapidly  rotated  the  corpuscles  are  thrown  to  the  outer  end 
of  the  tube,  which  must  be  closed  of  course.  The  volume  oc- 
cupied by  the  corpuscles  compared  with  the  original  blood 
volume  may  be  easily  seen. 

Koppe's  Hematocrit.  An  instrument  in  which  such  an 
observation  may  be  accurately  and  easily  made  was  devised 
by  Hedin  and  called  the  hematocrit.  A  special  form  of  this 
apparatus  was  constructed  by  Koppe  and  is  used  for  the  pur- 
pose of  comparison  of  corpuscle  volumes.  The  essential  part 
of  the  apparatus,  as  shown  in  the  figure,  is  a  graduated  capil- 
lary pipette  about  7  cm.  in  length,  which  may  be  closed  at  both 
ends  by  small  metal  plates.  At  the  upper  end  the  capillary 
bore  is  widened  out  so  as  to  form  a  small  mixing  vessel.  The 
pipette  proper  has  a  graduation  of  100  divisions.  By  means 
of  a  syringe  attached  to  the  pipette  by  a  bit  of  rubber  tubing 
blood  may  be  drawn  up  into  the  capillary  and  its  volume  accu- 
rately noted.  The  pipette  may  be  closed  and  rotated  now  rap- 
idly in  the  centrifugal  machine,  which  throws  the  corpuscles  to 
the  outer  end.  To  prevent  coagulation  it  is  best  to  moisten 
the  pipette  first  with  a  layer  of  cedar  oil  which  does  not  inter- 
fere with  the  reading  of  the  blood  volume.  The  relation  be- 
tween blood  volume  and  corpuscle  volume  may  thus  be  read 
off  on  the  graduation.  A  drop  of  similar  fresh  blood  is  next 
drawn  into  the  capillary  and  its  volume  noted ;  following  this 
some  solution  is  drawn  in  also,  and  then  with  the  blood  up  into 
the  wider  mixing  part,  where  by  means  of  a  bright,  fine  wire 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION. 


257 


the  two  liquids  may  be  stirred  together.  The  plates  are  then 
put  on  the  ends  of  the  pipette,  where  they  are  held  by  springs. 
The  pipette  may  be  rotated  as  before  in  the  centrifugal  ma- 
chine, the  rotation  being  continued  until  the  volume  occupied 
by  the  corpuscles  becomes  constant.  By  using  a  number  of 
pipette  tubes  it  is  possible  tO'  employ  different  mixtures  and 


Fig.  25.  The  essential  part  of  the  Koeppe  hematocrit.  The  measuring  tube 
a  is  closed  by  two  plates,  b  and  c,  which  are  held  fast  by  the  springs  d.  The 
tube  is  filled  by  means  of  a  peculiar  syringe  shown  at  the  right. 

soon  find  one  in  which  the  corpuscle  volume  remains  normal. 
If  a  series  of  sugar  or  salt  solutions  of  known  osmotic  pres- 
sure are  employed,  that  of  the  blood  must  be  taken  as  equiva- 
lent to  the  pressure  in  the  solution  for  which  no  change  in  the 
volume  of  the  corpuscles  occurs. 

Conversely  the  apparatus  may  be,  and  is,  frequently  employed  to  find 
osmotic  pressures  of  solutions.  The  volume  of  the  corpuscles  is  found 
in  some  solution,  of  cane  sugar  for  example,  which  has  about  the  same 
osmotic  pressure  as  the  blood,  but  which  must  be  accurately  known. 
Then  other  solutions  of  a  new  substance  are  tested  until  two  are  found 
which  give  volumes,  one  greater  and  the  other  less  than  that  with  the 
sugar.  A  simple  calculation  will  then  give  the  concentration  of  the  solu- 
tion of  the  substance  under  comparison  which  has  the  same  osmotic 
pressure  as  the  standard  sugar  solution.  The  method  would  naturally 
fail  for  any  substance  which  acts  chemically  on  the  blood  or  which  de- 
stroys the  corpuscles,  such  as  urea  or  glycerol. 
18 


258  PHYSIOLOGICAL    CHEMISTRY. 

CLINICAL   USES   OF   THE   HEMATOCRIT. 

On  the  assumption  tliat  tlie  volume  occupied  by  the  corpus- 
cles \-aries  with  the  num1)er  of  cells,  attempts  have  been  made 
to  use  the  hematocrit  in  place  of  the  cell  counter.  With  nor- 
mal blood  cells  the  relation  is  practically  constant  and  a  volume 
of  50  per  cent  in  the  hematocrit  corresponds  very  closely  to 
the  average  5.000.000  cells  per  cubic  millimeter.  But  unfor- 
tunately where  such  a  simple  method  of  making  a  blood  cell 
count  is  the  most  desirable  it  is  at  the  same  time  the  least  reli- 
able, since  in  disease  the  corpuscles  do  not  always  retain  their 
normal  size.  A  factor  of  perhaps  greater  importance,  how- 
ever, is  obtained  by  taking  the  ratio  of  the  volume  as  found 
by  the  hematocrit  to  the  corpuscle  count  as  made  by  a  hemo- 
cytometer.  With  undiluted  blood  the  hematocrit  may  be  used 
to  determine  whether  or  not  pigmentation  has  taken  place.  If 
the  corpuscles  are  intact  a  nearly  colorless  serum  is  secured; 
a  more  or  less  reddish  serum  points  to  disintegration  of  the 
corpuscles. 

THE  ELECTRICAL  CONDUCTIVITY  OF  BLOOD. 

Electrolytes.  It  has  been  found  by  experiment  that  cer- 
tain solutions  conduct  the  electric  current  while  others  do  not. 
Pure  liquids  do  not  conduct  at  all.  as  a  rule.  Thus  absolutely 
pure  water,  glycerol,  alcohol,  anhydrous  sulphuric  acid  and 
similar  substances  are  practically  non-conductors.  Solutions 
of  many  organic  substances  are  likewise  non-conductors,  prac- 
tically. The  sugars,  for  example,  belong  to  this  class.  But 
organic  acids  and  salts  and  many  so-called  basic  bodies  are, 
like  the  corresponding  inorganic  substances,  conductors.  In 
general,  liquid  conductors  or  electrolytes  are  compounds  wdiich 
in  solution  separate  or  dissociate  into  component  parts  or  ions 
more  or  less  perfectly.  The  mineral  salts  and  inorganic  acids 
and  alkalies  are  in  general  good  conductors,  as  they  "  ionize  " 
to  a  considerable  degree. 

Blood  serum  has  the  power  of  conducting  the  current  and 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION. 


259 


mainly  because  of  its  content  of  salts.  The  proteins  in  abso- 
lutely pure  condition,  salt  free,  are  probably  non-conductors. 
Some  of  them,  however,  because  of  their  acid  character  exist 
in  combinations  resembling  salts  and  these  have  a  weak  con- 
ducting power.     But,  because  of  their  high  molecular  weight, 


Fig.  26.  Diagram  of  Wheatstone  bridge  connections.  A  represents  a  cell  or 
induction  coil,  ac  the  bridge  wire,  5'  the  standard  resistance  with  which  com- 
parison is  made,  R  the  conductivity  cell  containing  the  substance  under  ex- 
amination. In  most  conductivity  experiments  ^  is  a  small  induction  coil,  a 
telephone,  as  shown,  being  employed  as  the  current  indicator. 

the  part  which  this  conductivity  plays  in  the  total  conductivity 
of  the  blood  is  very  small.  As  applied  to  the  blood,  therefore, 
conductivity  measurements  give  us  an  idea  of  the  number  of 
salt  molecules  present,  or  inorganic  concentration. 


Fig.  27.  Simple  Wheatstone  bridge.  The  bridge  wire  of  exact  length  is 
stretched  over  the  graduated  scale.  The  several  wire  connections  are  made  at 
the  binding  posts  lettered. 

In  practice  conductivity  is  the  reciprocal  of  resistance,  which  is  the 
factor  actually  measured.  Resistance  is  expressed  in  terms  of  some 
standard  arbitrarily  chosen,  and  comparisons  are  usually  with  the  "  ohm  " 
as  the  final  standard.  The  legal  ohm  is  the  resistance  of  a  column  of 
pure  mercury  106.3  cm.  long  and  i  mm.  in  section  at  a  temperature  of 
0°,  but  for  practical  use   resistance   standards  of  wire   are   employed.     A 


260  PHYSIOLOGICAL    CHEMISTRY. 

series  of  standard  wire  resistances  running  from  a  tenth,  or  hundredth 
of  an  ohm  even,  to  looo  ohms  or  more  is  generally  employed  in  the  form 
of  a  resistance  "  set  "  or  "  box." 

In  dealing  with  solutions  the  unit  of  conductivity  is  taken  as  the 
reciprocal  of  the  resistance  of  a  substance  which,  in  the  form  of  a  column 
I  cm.  square  and  i  cm.  long  (a  symmetrical  cubic  centimeter),  has  a 
resistance  of  i  ohm.  That  is,  the  conductivity,  f,  is  measured  in  terms 
of  that  of  an  ideal  liquid,  one  symmetrical  cubic  centimeter  of  which  has 
a  conductivity  of  i  between  opposite  faces,  or  which  offers  between  the 
same  faces  a  resistance  of  i  ohm.  The  resistance  of  liquids  is  always 
found  in  small  vessels  of  glass  made  in  different  shapes  and  sizes  accord- 
ing to  the  character  of  the  liquid.  Small  platinum  plates  are  mounted 
in  the  vessels  and  it  is  the  resistance  of  the  column  between  these  which 
is  measured.  Before  use  the  resistance  capacity  of  the  vessel  must  be 
found.  This  is  done  by  measuring  in  it,  with  the  plates  in  fixed  position, 
the  resistance  of  some  liquid  the  conductivity  of  which  has  been  previously 
determined  by  some  standard  method.  The  data  for  several  solutions 
have  been  very  accurately  determined  and  are  everywhere  used  for  pur- 
poses of  graduation  of  conductivity  vessels.  With  such  a  standard  liquid 
with  conductivity  k  we  find  in  our  cell  the  resistance,  R.  The  resistance 
capacit}',  C,  is  given  by  the  relation  : 

C  =  Rk. 

That  is,  C  is  the  resistance  which  would  be  found  in  the  vessel  if  it  were 
filled  with  a  liquid  of  unit  conductivity,  and  is  used  as  a  constant  in  all 
following  calculations  with-  the  same  vessel  when  we  wish  to  find  «.  R 
we  always  find  by  direct  measurement  in  ohms  and  with  C  known  we 
have  now : 

C 
''=R- 

The  resistance  of  liquids  cannot  be  found  as  is  that  of  a  solid  by  means 
of  the  Wheatstone  bridge  combination  and  a  galvanometer,  since  under 
such  circumstances  liquids  suffer  hydrolysis  with  rapid  change  of  resist- 
ance. In  place  of  the  direct  current  and  galvanometer  Kohlrausch  sug- 
gested the  use  of  a  weak  induction  current,  with  a  telephone  as  current 
indicator.  With  this  arrangement,  which  is  illustrated  by  the  annexed 
diagram,  it  is  possible  to  measure  the  conductivity  of  the  serum  or  other 
liquid  very  readily;  a  simple  form  of  Wheatstone  bridge  is  shown  also. 

ac  represents  the  graduated  wire  of  the  Wheatstone  bridge,  5"  the  stand- 
ard resistance  with  which  comparison  is  made,  R  the  cell  containing  the 
serum  or  other  liquid  under  investigation,  T  the  telephone  which  ceases 
to  buzz  when  no  current  passes  through  bd.  This  gives  the  "  null "  point 
in  the  combination  and  when  this  is  found  the  following  proportion  holds : 

ab :  be : :  S :  R. 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION. 


261 


ab  and  be  are  read  off  directly  as  bridge  wire  lengths,  5"  is  the  known  com- 
parison resistance.     Hence  the  unknown  cell  resistance  is  given  by 


R  =  S 


ad 


As  6"  in  practice  is  always  taken  as  10,  100  or  1000  ohms  and  ac  is  always 
divided  decimally,  tables  are  constructed  giving  directly  the  value  of  R 
for  any  value  of  ab  read  off.  In  practice  the  cell  R  is  always  kept  at  a 
constant  temperature,   as  the  conductivity  of  liquids  varies  greatly  with 


Fig.    28.     Simple    form    of 
Kohlrausch   conductivity  cell. 


Fig.  29.  Conduc- 
tivity cell  for  poor 
conductors  or  smaller 
quantities. 


temperature  changes.  To  maintain  this  constant  temperature  the  cell  is 
usually  immersed  in  a  large  water  thermostat,  so  constructed  that  it  may 
be  readily  controlled.     Forms  of  cells  are  illustrated. 

The  electrical  conductivity  of  urine  is  also  an  important  facto/  which 
may  be  found  by  the  same  kind  of  apparatus,  and  which  will  be  discussed 
later.  The  arrangement  of  apparatus  for  such  work  is  shown  in  the 
adjoining  figure. 

Value  of  the  Conductivity  for  Blood,  Expressed  in  the 
terms  just  explained  the  value  of  the  conductivity  of  blood 
serum  is  about  «:  =  0.012,  or  expressed  in  another  form  very 
convenient  for  calculation  120X10"''.  A  good  part  of  this 
conductivity  is  due  to  the  sodium  chloride  present.  If  the 
chlorides  be  accurately  determined  by  one  of  the  usual  meth- 
ods of  quantitative  analysis  and  the  proper  conductivity  cor- 
responding to  this  salt  content  be  calculated,  which  is  possible 
with  a  considerable  degree  of  accuracy,  and  subtracted  from 
the  total  or  observed  conductivity  a   remainder  is  obtained 


262 


PHYSIOLOGICAL    CHEMISTRY, 


which   measures   the  "  achlorithc  "   conchictivity.    that   is   the 
conductivity  due  to  the  carl^onates  and  phosphates  present. 


The  conductivity  of  the  sahs  in  the  serum  is  somewhat  less 
than  in  pure  water,  but  it  is  possible  to  make  a  correction  for 


PHYSICAL    METHODS    IN    BLOOD    EXAMINATION.  263 

this  interference  of  the  proteins  and  obtain  satisfactory  values. 
The  conductivity  determination  coupled  with  a  few  simple 
chemical  tests  gives  probably  a  better  view  of  the  inorganic 
combinations  in  the  serum  than  would  be  found  by  an  exam- 
ination of  the  ash  of  the  blood,  since  the  ash  must  contain  sul- 
phur and  phosphorus  salts  resulting  from  the  oxidation  of  the 
organic  compounds  of  these  elements.  The  general  method 
of  calculating  conductivities  in  a  mixed  fluid  like  the  blood 
will  be  discussed  under  the  head  of  conductivity  of  the  urine. 
The  information  furnished  by  conductivity  measurements  is, 
it  will  be  seen,  an  extension  of  that  furnished  by  the  osmotic 
pressure  determinations.  By  a  combination  of  the  two  proc- 
esses it  is  possible  to  distinguish  approximately  between  the 
concentrations  of  several  classes  of  molecules  present,  and  to 
follow  variations  in  these  concentrations  rapidly.  As  yet  the 
clinical  value  of  the  method  is  somewhat  uncertain,  however. 


CHAPTER    XIV. 

SOME  SPECIAL   PROPERTIES   OF  BLOOD  SERUM.     BACTERI- 
CIDAL   ACTION.      PRECIPITINS.    AGGLUTININS, 
BACTERIOLYSINS,  HEMOLYSINS. 

SELF  PRESERVATION  OF  THE  BLOOD. 

In  earlier  experiments  on  transfusion  of  blood  to  supply  a 
loss  brought  about  by  excessive  bleeding  it  was  recognized 
that  the  added  blood  sometimes  seemed  to  act  as  a  poison  to 
the  individual  to  whom  it  was  given.  It  was  found  later  that 
this  toxic  action  followed  the  passage  of  blood  from  one  species 
of  animal  to  another,  but  that  the  transfusion  from  man  to 
man.  from  dog  to  dog  or  from  rabbit  to  rabbit  was  not  accom- 
panied by  the  same  danger.  Such  observations  were  fre- 
quently made  and  gradually  led  to  the  conclusion  that  the 
plasma  or  serum  of  the  blood  of  each  animal  contains  a  some- 
thing which  has  a  destructive  action  on  the  corpuscles  of  other 
bloods  and  which  may  be  designed  to  protect  the  blood  from 
the  action  of  any  foreign  substance.  Various  theories  have 
been  put  forward  to  explain  this  recognized  property  of  the 
serum.  As  yet  our  knowledge  in  this  field  is  largely  of  the 
empirical  order,  and  scarcely  suitable  for  clear  elementary 
presentation.  But  the  importance  of  the  subject  as  thus  far 
developed  is  so  great  that  a  short  chapter  on  what  seems  most 
satisfactorily  established  may  not  be  out  of  place.  The  phe- 
nomena in  question  are  certainly  chemical  and  from  this  side 
must  receive  their  final  explanation.  Numerous  related  phe- 
nomena are  found  to  call  for  the  same  kind  of  consideration. 

It  has  long  been  known  that  the  large  white  cells  of  the 
blood,  the  so-called  leucocytes,  have  the  peculiar  power  of 
destroying  bacteria  or  other  foreign  cells  which  find  their  way 
into  the  blood  stream.  Hence  these  corpuscles  have  been 
called   phagocytes   or   devouring   cells.     This    destruction    of 

264 


SPECIAL  PROPERTIES  OF  BLOOD  SERUM.         265 

small  invading  organisms  they  seem  to  accomplish  by  a  kind 
of  digestive  process  which  in  a  general  way  may  be  followed 
under  the  microscope.  To  some  extent  the  destruction  of  one 
kind  of  blood  by  another  may  possibly  be  accounted  for  in  this 
way.  But  the  chief  action  is  certainly  of  a  different  character. 
While  the  white  cells  are  in  a  measure  protective  agents,  active 
in  destroying  elements  which  would  be  harmful  if  left  in  the 
blood,  it  seems  altogether  likely  that  the  most  important  con- 
serving forces  in  the  blood  are  soluble  compounds,  possibly  of 
the  nature  of  enzymes.  This  view  has  been  gradually  devel- 
oped and  rests  on  a  basis  of  experiment  and  observation. 

Harmful  foreign  bodies  entering  the  blood  may  be  in  the 
nature  of  cells,  as  of  bacteria,  or  they  may  be  the  poisons  called 
toxins  produced  by  bacteria.  Anything  in  the  blood  which 
resists  or  overcomes  the  force  of  this  invasion  is  called  an  anti 
body.  Normal  serum  seems  to  contain  a  number  of  anti  sub- 
stances, which  have  received  different  names,  depending  on 
how  or  against  what  they  act.  Some  are  called  precipitins, 
others  agglutinins,  cytotoxins,  etc.,  which  terms  will  be  ex- 
plained later.  In  addition  tO'  the  anti  bodies  normally  present 
in  sera  in  variable  amounts,  and  which  confer  a  certain  degree 
of  immunity,  there  may  be  produced  artificially  a  greatly  in- 
creased specific  immunity  against  some  particular  invasion. 
It  was  the  discovery  of  this  fact  which  in  reality  led  to  the 
systematic  study  of  the  whole  phenomenon. 

The  castor  oil  bean  contains  a  peculiar  poisonous  principle 
known  as  ricin,  which  if  given  in  relatively  large  doses  is  fatal, 
but  against  which  an  animal  may  be  immunized  by  treatment 
with  gradually  increasing  small  doses.  An  experiment  made 
by  Ehrlich,  to  whom  much  is  due  in  this  field  of  investigation, 
showed  that  the  serum  of  the  treated  animal  must  contain  now 
a  specific  anti  body  capable  of  neutralizing  the  physiological 
action  of  ricin.  He  found  that  if  the  ricin  poison  and  the 
serum  of  the  immunized  animal  are  mixed  in  vitro  in  certain 
proportion,  and  then  injected  into  a  fresh  non-immunized  ani- 
mal no  toxic  action  follows.     The  serum  of  the  first  or  im- 


266  PHYSIOLOGICAL    CHEMISTRY. 

munized  animal  has  acquired  the  property  of  chemically  com- 
bining with  or  in  some  manner  neutralizing  the  action  of  the 
poison.  That  something  akin  to  a  chemical  action  is  here  in 
question  is  shown  by  the  fact  brought  out  by  further  experi- 
ments that  certain  proportions  must  be  observed  in  the  mixing 
of  the  serum  and  toxic  substance  just  as  in  the  complete  neu- 
tralization of  an  acid  by  an  alkali.  It  was  further  found  that 
this  combination  may  be  hastened  by  heat  and  retarded  by 
cold,  which  is  true  of  most  chemical  reactions.  The  behavior 
is  also  specific ;  that  is,  the  animal  immunized  against  the  cas- 
tor bean  poison  is  not  immunized  thereby  against  other  veget- 
able toxic  substances,  as  abrin  for  example,  and  the  serum  of 
the  animal  will  not,  in  vitro,  neutralize  the  toxic  abrin  solution. 
The  same  general  condition  has  been  recognized  in  connec- 
tion with  other  immunizations  and  the  characteristically  spe- 
cific nature  of  the  anti  body  produced  in  the  serum  has  been 
shown  beyond  question.  The  anti  bodies  protective  against 
diphtheria  have  no  effect  against  the  toxins  of  tetanus  or  other 
disease  and  vice  versa.  With  such  facts  established,  inquiry 
was  naturally  directed  toward  the  question  of  the  chemical 
nature  of  these  substances  and  to  the  question  of  their  mode 
of  action.  In  the  voluminous  discussions  which  have  been 
carried  on  over  these  points  it  is  not  always  easy  to  distinguish 
between  observed  facts  and  stoutly  maintained  theories. 

GENERAL    CHARACTER   OF    THE   ANTI   BODIES. 

Antitoxins  Proper.  Foreign  harmful  agents  gaining  ac- 
cess to  the  blood  may  be  of  several  kinds.  Some  of  these  are 
soluble  toxic  compounds,  products  of  cell  action,  which  in 
their  behavior  bear  some  relation  to  strong  alkaloidal  poisons. 
Many  of  these  toxins  are  produced  by  bacteria  in  the  animal 
body  during  the  progress  of  disease  and  the  symptoms  ob- 
served are  often  due  to  the  action  of  these  poisons  rather  than 
to  mechanical  disturbances  brought  alx>ut  by  the  bacteria  di- 
rectly. The  toxins  as  soluble  products  have  the  power  of 
wandering  with  the  blood  stream  and  thus  reaching  particu- 


SPECIAL  PROPERTIES  OF  BLOOD  SERUM.        26/ 

larly  vulnerable  or  susceptible  organs.  The  soluble  serum  con- 
stituent which  is  normally  present  in  small  amount  or  which 
may  be  developed  there  to  neutralize  the  toxin  in  some  man- 
ner is  called  an  antitoxin  in  the  restricted  sense.  There  is 
reason  to  believe  that  the  two  things  combine  with  each  other 
in  a  true  chemical  union  and  leave  a  soluble  inert  product. 

Precipitins.  The  serum  of  normal  blood  contains  constit- 
uents antagonistic  not  only  to  toxic  substances  but  to  other 
sera  as  well.  The  serum  of  one  animal  tends  to  precipitate 
or  render  cloudy  the  serum  of  another.  This  effect  may  be 
greatly  increased  by  a  kind  of  cultivation,  which  may  be  illus- 
trated in  this  way.  Rabbits'  blood  has  normally  some  antag- 
onism for  ox  blood,  but  if  sterile  ox  serum  be  injected  into  the 
rabbit,  intraperitoneally  or  intravenously,  beginning  with 
small  doses  and  increasing  through  a  number  of  days,  a  con- 
dition is  finally  reached  in  which  the  rabbit  blood  serum  shows 
a  very  strong  precipitating  power  for  ox  serum.  The  small 
amount  of  anti  body  in  the  rabbit's  blood  has  evidently  in- 
creased enormously  through  this  treatment.  The  organism 
through  the  attack  of  the  foreign  serum  gradually  develops  a 
protective  agent  which  acts  through  exclusion  or  precipitation. 
This  serum  constituent  is  called  a  precipitin. 

A  vast  number  of  experiments  have  been  made  in  this  field 
and  the  subject  has  importance  in  different  directions.  We 
recognize  not  only  the  normal  effort  of  the  blood  to  protect 
itself  in  this  way,  but  also  the  remarkable  power  of  develop- 
ment in  the  peculiar  anti  body  here  concerned.  From  another 
standpoint,  however,  the  phenomenon  has  assumed  even 
greater  importance  and  that  is  in  the  identification  of  blood. 
This  precipitin  reaction  like  the  others  is  specific  and  the  serum 
of  the  rabbit  immunized  with  ox  serum  will  react  only  with 
the  ox  serum.  But  precipitins  do  not  seem  to  be  formed  in  the 
blood  of  animals  which  are  closely  related.  The  serum  of  a 
rabbit  which  has  been  treated  with  pigeon  serum  will  not  react 
with  chicken  serum;  an  anti  rabbit  serum  cannot  be  secured 
by  treating  a  guinea-pig  with  the  serum  of  rabbit's  blood. 
These  general  facts  have  been  confirmed  by  many  observations. 


268  PHYSIOLOGICAL    CHEMISTRY. 

Blood  Tests  with  Serum.  The  method  of  utilizing  these  generaliza- 
tions is  essentially  this.  Rabbits  are  the  animals  commonly  used  for  ex- 
periments, since  they  bear  the  treatment  in  general  well  and  yield  a  fairly 
large  quantity  of  immunized  blood  later.  Each  rabbit  is  treated  by  injec- 
tion with  the  blood  serum  of  one  animal,  these  injections  being  repeated 
a  number  of  times,  through  several  weeks.  Then  the  rabbit  is  killed, 
bled  and  the  blood  allowed  to  stand  for  separation  of  clot.  The  clear 
serum  is  preserved  in  sealed  tubes  for  future  use,  sometimes  with,  some- 
times without  addition  of  an  antiseptic,  as  putrefaction  does  not  appear 
to  impair  the  reaction.  By  immunizing  rabbits  separately  with  the  blood 
of  man,  the  ox,  horse,  pig,  dog,  sheep,  goat,  chicken,  etc.,  a  whole  series 
of  test  auti  sera  will  be  obtained,  with  which  it  is  possible  tp  identify 
most  of  the  common  bloods.  Not  much  blood  is  required  in  the  tests. 
A  few  drops  of  blood,  from  a  dried  clot  for  example,  is  soaked  in  water 
or  normal  salt  solution,  the  liquid  obtained  filtered  to  clarify  it  and  treated 
in  a  small  test-tube  with  two  or  three  drops  of  the  test-serum.  The  liquid 
to  be  tested  need  not  be  strong.  In  practice  it  should  be  divided  into  a 
number  of  small  portions  in  test-tubes  and  each  portion  should  receive 
a  few  drops  of  an  immunized  rabbit  serum.  Precipitation  or  clouding 
will  occur  in  the  tube  to  which  the  corresponding  anti  serum  is  added. 
For  example,  if  the  original  clot  of  blood  was  human  blood  the  extracted 
dilute  serum  in  all  the  tubes,  except  the  one  to  which  rabbit  blood  im- 
munized with  human  blood  was  added,  will  remain  clear;  other  tubes  with 
portions  of  the  e.xtracted  clot  show  no  reaction  with  the  few  drops  of 
rabbit  sera  immunized  with  the  blood  of  other  animals. 

The  medico-legal  importance  of  this  reaction  has  already  been  recog- 
nized and  tested  in  many  ways.  The  blood  of  certain  monkeys  seems 
to  react  as  does  human  blood,  but  those  who  have  practiced  the  test  most 
testify  as  to  its  certainty  and  wide  applicability  in  distinguishing  between 
human  blood  and  the  blood  of  the  common  domestic  animals. 

The  Cytotoxins.  This  name  is  given  to  certain  anti  com- 
pounds in  blood  which  are  destructive  of  form  elements.  The 
anti  bodies  before  considered  deal  with  soluble  substances,  but 
here  we  have  to  consider  something  whose  power  extends  to 
the  breaking  down  of  cell  structures,  whether  of  the  blood  cor- 
puscle or  of  bacteria.  In  the  one  case  the  term  hemolysin  is 
used  to  describe  the  anti  body :  in  the  other  case  the  term  hac- 
tcriolysiii  is  employed.  In  their  mode  of  action  these  agents 
appear  to  be  much  alike  and  both  are  found  in  normal  bloods. 
Both  also  may  be  greatly  increased  artificially. 

The  hemolytic  action  of  one  blood  on  another  was  first  ob- 
served in  experiments  on  blood  transfusion  which  have  been 


SPECIAL    PROPERTIES    OF    BLOOD    SERUM.  269 

referred  to  already.  A  foreign  blood  introduced  into  the  cir- 
culation of  an  animal  of  a  different  species  brings  about  a 
variety  of  changes ;  clots  are  sometimes  formed  and  from  re- 
sultant changes  in  pressure  serous  exudations  may  follow. 
Hemoglobinuria  is  a  general  consequence  and  this  of  course 
results  from  a  breaking  down  of  blood  corpuscles  in  quantity. 
It  has  been  assumed  by  some  writers  that  this  hemolytic 
effect  is  possibly  due  to  altered  osmotic  pressure  in  the  blood, 
as  similar  phenomena  are  brought  about  by  the  admixture  of 
blood  with  weak  solutions.  But  the  peculiar  specificity  of 
artificial  hemolysis  shows  that  this  explanation  is  not  satis- 
factory. 

If  the  blood  of  man,  for  example,  receives  an  injection  of 
human  blood  under  proper  condition  no  harm  results,  but  if 
ox  blood  be  used  the  case  is  different.  A  large  transfusion  of 
the  ox  blood  might  be  at  once  fatal,  the  hemolysins  of  that 
destroying  the  human  corpuscles.  On  the  other  hand  trans- 
fusion of  small  amounts  of  ox  blood  would  have  different 
effects  varying  with  the  manner  of  transfusion.  With  but 
little  ox  blood  added  the  human  hemolysins  would  be  greatly 
in  excess  and  by  their  chemical  mass  action  would  bring  about 
a  relatively  great  destruction  of  the  ox  blood  corpuscles,  while 
the  corpuscles  of  the  human  blood  would  suffer  but  little 
change.  But  more  than  this  would  probably  take  place  as 
illustrated  by  what  has  been  observed  with  certain  lower  ani- 
mals. The  serum  of  the  eel  is  especially  destructive  of  the 
corpuscles  of  rabbit's  blood  and  a  large  injection  of  eel  serum 
into  the  rabbit  would  produce  death.  With  repeated  small 
doses,  however,  the  rabbit's  blood  is  stimulated  to  develop  the 
antitoxin  or  antihemolysin  which  protects  against  the  eel 
serum  poison.  A  condition  of  immunity  is  thus  reached,  and 
what  has  been  shown  for  the  rabbit  has  been  shown  for  other 
animals.  An  explanation  of  the  origin  of  the  hemolysins  will 
be  offered  below,  but  now  we  are  concerned  only  with  the 
fact. 

In  the  above  cited  experiment  the  eel  serum  develops  gradu- 


270  PHYSIOLOGICAL    CHEMISTRY, 

allv  an  antibemolysin  which  works  to  prevent  further  destruc- 
tion of  the  rabbit  corpuscles.  But  the  action  does  not  stop 
here.  By  injection  of  blood,  hemolysins  for  the  corpuscles  of 
this  blood  are  also  formed.  Numerous  experiments  of  this 
kind  have  been  made  with  animals.  For  example,  when  rab- 
bit's blood  is  gradually  injected  into  the  dog  the  production  of 
hemolysins  is  stimulated  and  the  serum  of  the  dog  so  treated 
is  found  to  be  far  more  toxic  for  the  rabbit  than  was  the  orig- 
inal serum.  This  toxic  action,  by  test-tube  experiments,  has 
been  found  to  be  parallel  to  the  hemolytic  action  of  the  dog 
serum  on  the  rabbit  corpuscles,  thus  showing  that  the  toxicity 
may  depend  on  the  destruction  of  the  corpuscles.  The  hemo- 
lysins produced  as  just  explained  are  also  in  general  specific  in 
their  character,  which  can  be  followed  by  experiments  in  vitro 
as  well  as  in  corporc. 

In  general  the  bactericidal  action  of  serum  resembles  its 
hemolytic  action,  although  control  experiments  in  vitro  can- 
not be  as  readily  performed.  We  have  therefore  the  hactcri- 
olysins  to  consider  along  with  the  other  cell  destroyers.  These 
bodies  exist  to  some  extent  in  normal  blood  and  other  body 
fluids  and  serve  to  protect  the  organism  against  the  attack  of 
bacteria  which  in  any  way  gain  admission  to  the  body.  Milk 
is  relatively  rich  in  bacteriolysins  and  hence  the  well-known 
germicidal  action  which  has  been  long  recognized.  In  this 
respect  the  behavior  of  mother's  milk  is  more  marked  than  that 
of  cow's  milk.  Besides  the  cytotoxins  of  this  class  normally 
present  in  blood,  specific  bodies  may  be  developed  by  the  gen- 
eral methods  followed  in  other  cases,  that  is  by  the  gradual 
introduction  of  cultures  of  specific  bacteria,  beginning  with 
cultures  of  relatively  little  virulence.  In  this  way  the  blood 
of  the  treated  animal  becomes  immune  for  some  one  bacterium 
species  and  develops  the  power  of  destroying  that  bacterium 
only  for  which  it  was  specially  immunized.  The  same  animal 
may  be  immunized  against  several  kinds  of  bacteria  at  the 
same  time  and  the  different  specific  bacteriolysins  do  not  ap- 
pear to  have  any  destructive  action  on  each  other.     They  exist 


SPECIAL    PROPERTIES    OF    BLOOD    SERUM.  2/1 

together  in  the  blood  just  as  the  different  proteins  may  exist 
side  by  side. 

Through  the  process  of  immunization  the  blood  of  the  ani- 
mal acquires  not  only  the  power  of  attacking  the  specific  bac- 
terium, but  also  the  toxins  of  this  bacterium.  At  least  two 
kinds  of  anti  bodies  are  therefore  produced  and  there  are  con- 
ditions in  which  only  one  of  these  may  be  active.  A  serum 
may  be  active  in  the  breaking  down  of  bacterial  cells,  but  inert 
as  against  the  poisons  produced  by  such  cells.  The  complexi- 
ties of  the  phenomena,  however,  cannot  be  detailed  here.  It 
should  be  said  further  that  bacteria  produce  hemolysins,  which 
are  probably  part  of  the  toxins  secreted.  At  any  rate  some  of 
the  toxins  found  in  cultures  are  strongly  hemolytic. 

Agglutinins.  Among  the  several  modes  of  defense  ob- 
served in  sera  of  various  animals  that  of  agglutination  of  in- 
vading cells  must  next  be  briefly  considered.  We  have  seen 
that  blood  cells  and  bacterial  cells  may  suffer  a  kind  of  dissolu- 
tion through  the  action  of  hemolysins  or  hacteriolysins,  and 
that  a  foreign  serum  is  attacked  by  the  precipitins.  In  addition 
to  these  defensive  anti  bodies  there  are  present  others  which 
work  by  agglutinating  or  precipitating  cells.  A  certain  simil- 
arity exists  between  these  bodies  and  the  precipitins,  but  inves- 
tigations appear  to  show  that  they  are  distinct.  The  agglutin- 
ating power  is  found  in  normal  serum,  and  like  the  other  anti 
agencies  it  may  be  greatly  increased  artificially  and  by  the 
same  general  means.  Agglutinins  as  precipitating  agents  en- 
ter into  a  loose  kind  of  combination  with  the  cells  which  they 
throw  down.  There  is  here  a  suggestion  of  combination  in 
some  kind  of  chemical  proportions. 

Bacteria  agglutinins  and  blood  cell  agglutinins  are  to  some 
extent  specific,  but  apparently  less  so  than  are  the  precipitins. 
Because  of  this  specificity  the  phenomenon  has  been  applied  in 
a  method  of  diagnosis.  Following  observations  of  Gruber 
and  others,  Widal  suggested  a  test  which  is  now  commonly 
employed  in  diagnosis  of  typhoid  fever.  It  is  essentially  this : 
Widal  Test.     A  small  amount  of  the  blood  or  serum  of 


2/2  PHYSIOLOGICAL    CHEMISTRY. 

the  suspected  typhoid  fever  patient  is  mixed  on  a  slide  with  a 
bouillon  culture  of  typhoid  bacilli.  After  a  time  the  mixture 
is  examined  with  the  microscope.  If  the  suspected  blood  con- 
tains the  agglutinins  developed  in  the  disease  "  clumping  "  or 
precipitation  of  the  bacteria  from  the  culture  must  follow.  (It 
is  held  as  characteristic  that  loss  of  motility  must  also  be  ob- 
served.) The  intensity  of  the  agglutinin  reaction  may  be 
estimated  l)y  noting  the  degree  of  dilution  in  which  the  Ijlood 
serum  will  still  agglutinate  the  bacteria  from  the  bouillon 
culture. 

Other  Anti  Bodies.  In  the  above  l)rief  survey  of  the  sub- 
ject of  anti  bodies  present  or  developed  in  the  blood  only  those 
which  have  been  the  object  of  most  frec[uent  investigations 
have  been  mentioned.  Bacteriologists  have  called  attention 
to  numerous  other  ^•arieties  or  subdivisions,  but  it  is  not  the 
purpose  of  this  chapter  to  take  up  the  discussion  of  details. 
What  has  been  given  is  sufficient  to  call  attention  to  the 
broader  principles  concerned. 

CHEMICAL    NATURE    OF    THE    ANTI    BODIES. 

On  this  topic  much  has  been  written,  but  as  yet  no  satisfac- 
tory answer  can  be  given  to  the  question  :  What  are  they  chem- 
ically? As  formed  in  the  serum  of  blood  or  in  milk  it  may 
reasonably  be  assumed  that  they  must  bear  some  relation  in 
comi^osition  to  the  protein  bodies.  On  this  basis  attempts  have 
•  been  made  to  separate  them  by  fractional  precipitation  reac- 
tions such  as  were  developed  by  Hofmeister  and  others  for  the 
proteins  and  which  have  been  detailed  in  former  chapters. 

It  appears  from  the  evidence  thus  far  offered  that  some  of 
these  bodies,  at  least,  must  be  classed  among  the  globulins. 
Pick  and  others  have  recently  been  able  to  separate  the  active 
substances  from  several  kinds  of  immune  sera  and  establish 
pretty  accurately  the  limits  of  precipitation.  The  active  frac- 
tions separated  contained  the  real  anti  bodies  in  minute  amount 
only,  probably.  In  some  cases  they  were  found  in  the  euglob- 
ulin  fraction,  and  in  other  cases  in  the  pseudoglobulin  fraction 
of  the  precipitate. 


SPECIAL    PROPERTIES    OF    BLOOD    SERUM.  2/3 

There  has  been  much  speculation  as  to  the  part  of  the  blood 
which  gives  rise  to  these  various  anti  bodies.  They  are  solu- 
ble and  may  not  be  separated  by  filtration,  but  on  dialysis  they 
behave  as  other  substances  of  very  high  molecular  weight.  In 
many  respects  they  resemble  highly  active  proteolytic  ferments 
or  enzymes  as  the  characteristic  phenomena  are  exhibited  even 
in  dilutions  of  the  active  serum  of  i  :  20,000,  and  heat  and 
chemical  reagents  interfere  with  the  active  properties  much  as 
in  the  case  of  the  enzymes.  But  there  are  apparently  some 
exceptions  which  have  led  certain  authors  to  deny  their  en- 
zyme-like character.  From  the  sum  of  the  facts  observed 
in  the  occurrence  and  action  of  the  anti  bodies  several  writers 
have  been  led  to  think  of  them  as  derived  from  the  breaking 
down  of  the  highly  complex  polynuclear  white  corpuscles  of 
the  blood.  The  behavior  of  these  in  the  "  living  "  condition 
has  been  already  referred  to;  in  their  disintegration  it  is  pos- 
sible they  may  give  off  more  and  more  of  the  groups  on  which 
their  activity  depends.  But  there  are  other  possibilities  and 
these  will  be  referred  to  in  the  following  section. 

origin  and  mode  of  action  of  the  anti  bodies.   ehrlich's 

theory! 

In  the  early  days  of  observations  on  blood  serum  immunity 
the  doctrine  of  the  phagocytes  received  considerable  attention. 
With  increase  of  knowledge  this  theory  was  seen  tO'  be  inade- 
C[uate  to  account  for  accumulating  facts,  and  the  assumption 
of  the  soluble  proteolytic  ferments,  the  alexins,  was  next  to 
attract  attention.  These  may  be  formed  from  the  polynuclear 
leucocytes  or  more  remotely  in  the  organs  where  these  cells 
may  have  their  origin,  in  the  spleen,  for  example,  and  in  the 
marrow  of  bones.  As  the  name  indicates  the  alexins  are  pro- 
tective substances,  but  the  simple  assumption  of  these  bodies 
acting  alone,  as  a  chemical  reagent  would  for  example,  in  the 
annihilation  of  intruding  bacteria  was  soon  seen  to  be  too  nar- 
row to  accord  with  experience.  The  phenomenon  of  immu- 
nity through  the  alexins  or  other  bodies  is  a  complex  one,  but 
19 


2  74  PHYSIOLOGICAL    CHEMISTRY. 

lias  received  much  elucidation  through  numerous  ol-)servations 
of  recent  years.  One  of  the  most  important  of  these  is  con- 
cerned with  tlie  so-called  Pfeiffer  experiment. 

Pfeiffer's  Phenomenon.  In  his  famous  experiments  on 
the  behavior  of  cholera  bacteria  Pfeiffer  found  that  the  serum 
of  an  animal  which  had  been  immunized  against  cholera,  when 
tested  /;/  z'itro  against  the  vibrios,  seemed  deficient  in  bacteri- 
olytic power,  possessing  no  greater  activity,  evidently,  than 
that  due  to  the  proteolytic  ferment  of  the  normal  serum.  Ac- 
tivity may  be  given  to  the  immunized  serum,  however,  by  in- 
jecting it  back  into  the  peritoneal  cavity  of  the  animal ;  cholera 
vibrios  injected  at  the  same  time  are  quickly  destroyed.  The 
action  is  strictly  specific,  since  typhoid  bacilli  injected  in  the 
same  way  remain  active.  It  was  found  also  that  the  same 
result  can  be  reached  with  the  vibrios  by  adding,  in  vitro,  fresh 
normal  serum  to  the  latent  immunized  serum.  The  vibrios 
succumb  as  they  did  /;/  corporc,  showing  that  a  vital  action  is 
not  in  play.  Numerous  similar  observations  were  made  by 
Bordet  and  others.  From  all  these  experiments  it  is  evident 
that  at  least  two  things  are  concerned  in  the  bacteriolytic  action 
and  the  analogous  hemolytic  phenomena.  The  immune  body 
developed  in  the  course  of  immunization  does  its  work  as  a 
cell  destroyer,  whether  a  blood  corpuscle  or  a  bacterium,  only 
by  the  aid  of  something  normally  present  in  fresh  serum.  The 
specific  immune  body  was  found  to  be  thermostable,  that  is  it 
withstands  a  temperature  of  55°-70°  without  loss  of  its  spe- 
cial properties.  If  after  being  warmed  to  this  extent  the  im- 
mune serum  is  cooled  to  below  55°  and  mixed  with  fresh  nor- 
mal serum  the  full  cytolytic  activity  appears.  On  the  other 
hand  the  ferment  in  the  normal  serum  is  thermolabile;  a  tem- 
perature of  55°  or  above  destroys  it  permanently.  For  this 
thermolabile  normal  ferment  Ehrlich  proposed  the  name  addi- 
iiicuf.  This  is  the  same  as  the  alexin  body.  The  term  com- 
plement is  also  used  to  describe  the  same  thing  which  seems 
necessary  to  make  the  specific  immune  body  really  active. 

The  Side  Chain  Theory.     Thus  far  we  have  been  con- 


SPECIAL  PROPERTIES  OF  BLOOD  SERUM,        2/5 

cerned  with  the  results  of  experiments,  with  facts  about  which 
there  cannot  be  much  question.  But  a  comprehensive  theory 
to  correlate  all  these  generalizations. became  necessary.  Many 
attempts  have  indeed  been  made  to  establish  a  theoretical  basis 
for  the  doctrines  of  immunity,  but  it  remained  for  Ehrlich  to 
suggest  something  which  is  really  tangible  from  the  chemical 
standpoint.  To  follow  the  Ehrlich  notions  some  other  mat- 
ters must  be  explained  first. 

Years  ago  in  attempting  to  explain  some  of  the  properties 
of  large  organic  molecules,  Pasteur  introduced  the  notion  of 
molecular  asymmetry  into  chemical  science.  He  showed  the 
value  of  the  notion  of  configuration  in  dealing  with  certain 
classes  of  chemical  problems.  This  general  idea  was  greatly 
advanced  by  Emil  Fischer  in  his  papers  on  the  chemistry  of 
the  sugars.  Certain  ferments  were  found  to  decompose  one 
sugar  of  an  isomeric  group,  but  to  leave  the  other  almost  iden- 
tical sugars  untouched.  In  other  words  the  ferments  were 
found  to  observe  a  specific  selection,  and  to  work  as  ferments, 
the  enzymes  in  question  must  possess  a  certain  stereochemical 
structure  bearing  some  relation  to  the  stereochemical  structure 
of  the  sugar,  AVithout  this  relation  fermentation  cannot  take 
place.  In  order  to  make  his  meaning  plain  Fischer  employed 
a  figure  which  has  since  become  famous.  He  said,  in  speak- 
ing of  certain  glucosides,  "  Enzyme  and  glucoside  must  fit  into 
each  other  as  a  key  into  a  lock  in  order  that  the  one  may  be 
able  to  exert  a  chemical  action  on  the  other,"  In  one  of  his 
papers  Fischer  suggested  that  the  idea  of  related  molecular 
configuration  of  ferment  and  fermentable  body  may  prove  of 
value  in  physiological  investigations  as  well  as  in  chemistry 
and  in  the  development  of  the  theory  of  Ehrlich  this  predic- 
tion has  been  verified.  Toxins  and  anti  bodies  combine  with 
each  other  only  when  they  possess  corresponding  atom  groups, 
and  specificity  is  regarded  as  dependent  on  this  relative  con- 
figuration. 

Without  going  into  minute  details  the  chemical  part  of  the 
Ehrlich  theory  is  briefly  this.     Bacteria,  animal  cells  and  tox- 


2/6  PHYSIOLOGICAL    CHEMISTRY. 

ins  are  all  complex  aggregations  of  more  or  less  complex  mole- 
cules. The  latter  have  certain  configurations  dependent  on  the 
presence  of  side  chains  or  side  groups,  to  borrow  an  expres- 
sion from  organic  chemistry.  These  side  chains  are  directly 
or  indirectly  the  points  of  attack  or  defense  in  the  action  of 
the  several  bodies  on  each  other.  In  order  that  a  substance 
may  act  as  an  enzymic  poison  or  toxin  to  cells  of  the  body 
both  cells  and  toxins  must  therefore  possess  certain  reciprocal 
configurations.  It  has  been  suggested  further  that  these  side 
groups  are  concerned  in  all  the  actions  of  the  cells  and  that  it 
is  through  them,  for  example,  that  the  latter  absorb  their  nec- 
essary nutriment  and  elaborate  new  structures  from  it.  Some 
of  the  side  chains  may  be  constructed  to  combine  with  fats, 
some  with  carbohydrates  and  some  with  proteins,  but  in  the 
presence  of  toxins  or  bacteria  with  the  right  kind  of  side  chains 
combination  with  these  may  take  place  instead.  Many  of 
these  combinations,  perhaps  all,  take  place  not  directly,  but 
indirectly,  through  the  presence  of  an  intermediary  body  or 
group  which  itself  must  possess  two  linking  complexes  or 
groups  with  proper  configurations. 

To  describe  these  various  groups  certain  special  names  have 
been  suggested.  Iinmnnc  body  is  the  specific  substance 
formed  in  the  immunizing  process  against  cells  and  is  known 
also  by  several  other  names,  among  which  ajiiboccptor  and  iii- 
tcmicdiary  body  are  the  most  commonly  used.  The  coinplc- 
nicnt,  addimcnt  or  alexin  is  the  ferment-like  body  found  in 
normal  fresh  serum,  and  which  added  to  the  immune  body 
makes  up  the  real  cytotoxin.  It  is  not  specific  and,  as  inti- 
mated above,  is  sensitive  to  heat,  and  also  to  light  and  air 
(oxygen).  The  various  groups  of  the  large  cell  complex, 
whether  of  a  body  cell  or  of  a  bacterium,  which  have  the  power 
of  uniting  with  other  groups  are  called  receptors.  The  part 
of  the  receptor  which  is  free  to  combine  with  a  food  molecule 
or  analogous  substance  is  called  its  haptophorous  group.  Ehr- 
lich  pictures  the  part  played  by  the  immune  body  and  the  com- 
plement in  this  way.     Assuming  that  a  bacterial  cell  enters  a 


SPECIAL  PROPERTIES  OF  BLOOD  SERUM.         2// 

medium  where  these  complexes  are  present  the  immune  body 
attaches  itself  to  the  haptophorous  group  of  the  cell.  In  this 
condition  no  action  follows  immediately ;  but  the  immune  com- 
plex has  itself  two  haptophorous  groups  or  side  chains, 
through  one  of  which  the  union  with  the  bacterial  cell  is  ef- 
fected, while  with  the  other  it  joins  on  to  the  addiment  or 
complement  through  its  haptophorous  group.  In  this  way  the 
complement,  which  alone  is  inactive  or  unable  to  attack  the 
cell,  is  brought  into  the  immediate  neighborhood  of  the  latter, 
where  its  proteolytic  efforts  are  more  effective.  Ever}^  fresh 
normal  serum  seems  to  have  present  enough  of  the  complement 
groups;  the  question  of  destroying  the  invading  cell  depends 
then  on  the  number  of  immune  or  intermediary  groups  in  the 
field.  Another  part  of  the  Ehrlich  theory  attempts  to  account 
for  these. 

The  Immune  Group.  Ehrlich  traces  the  development  of 
the  immune  body  to  the  spontaneous  effort  on  the  part  of  the 
cell  to  protect  and  regenerate  itself  in  case  of  partial  destruc- 
tion. The  various  cells  of  the  body  exist  in  a  kind  of  equilib- 
rium with  each  other.  An  injury  to  one,  that  is  the  loss  of 
some  of  its  side  chains,  immediately  leads  to  an  effort  at  com- 
pensation. The  hyperplasia  observed  in  an  organ  may  extend 
to  the  single  cells  and  in  consequence  of  this  we  have  over- 
compensation. The  cell's  efforts  at  regeneration  lead  to  the 
production  of  more  side  chains  than  are  actually  necessary  and 
some  of  these  combine  with  the  aid  of  their  receptor  groups 
with  toxin  or  with  complement.  Many  are  formed  in  excess 
and  are  thrown  off  into  the  circulation.  These  free  receptops 
constitute  the  various  anti  or  immune  bodies.  Combining 
with  complement  groups  they  form  the  true  cytotoxins.  The 
larger  the  number  of  free  receptors  thrown  off  into  the  blood 
by  the  over-compensating  efforts  of  the  attacked  cells  the 
stronger  is  its  cytotoxic  or  antitoxic  character  since  these  re- 
ceptors hold  either  the  toxin  or  foreign  cell  and  thus  protect 
the  parent  native  cell  from  attack  or  destruction. 

The  fundamental  point  then  in  the  Ehrlich  theory  of  serum 


2/8  PHYSIOLOGICAL    CHEMISTRY. 

immunity  is  this  formation  of  side  chains  in  excess  by  over- 
compensation, and  is  founded  on  the  somewhat  earher  Weigert 
doctrine  of  cell  regeneration  and  over-compensation  in  general. 
Ehrlich  has  added  the  chemical  conceptions  of  side  chain 
groups  and  has  drawn  numerous  illustrations  from  organic 
chemistry  to  show  how  they  may  act.  Large  molecules  hold- 
ing amino,  sulphonic  acid  or  halogen  addition  groups,  for  ex- 
ample, may  lose  these  or  take  them  on  again  or  take  others 
like  them  without  losing  their  identity.  Reagents  acting  on 
such  a  large  molecule  attack,  not  the  nucleus  but  these  side 
chains  in  general.  The  simple  organic  molecule  has  not  the 
power  of  self  regeneration,  but  the  cell,  which  is  a  collection 
of  many  such  molecules,  has  the  power  of  forming  new  ma- 
terials from  the  nutritive  substances  furnished  to  it.  If  whole 
new  cells  are  formed  why  not  parts  of  cells  or  the  outlying 
side  groups  as  well,  and  this  is  the  Ehrlich  assumption,  which 
is  not  unreasonable. 

As  explained  above  these  side  chains  or  receptors  are  of 
various  kinds.  Three  distinct  types  or  orders  are  easily  rec- 
ognized. Receptors  of  the  First  Order  have  one  haptophor- 
ous  group  and  form  antitoxins.  That  is,  they  combine  chem- 
ically with  the  soluble  toxins  in  the  serum  and  in  a  sense 
neutralize  them.  Receptors  of  the  Second  Order  have  one 
haptophorous  group  with  which  a  foreign  molecule  or  group 
may  be  held  and  one  special  group  which  performs  the  func- 
tion of  an  agglutinin  or  precipitin.  Receptors  of  the  Third 
Order  or  amboceptors  have  two  haptophorous  groups  with 
which  two  things  may  be  united.  One  of  these  is  the  foreign 
cell  (through  its  corresponding  haptophorous  group)  and  the 
other  the  complement.  In  this  way  the  complement  or  alexin 
is  able  to  work  on  the  invading  cell  and  attack  it  through  its 
"zymotoxic"  group.  These  amboceptors  are  in  themselves  in- 
active and  can  behave  as  cytotoxins  (hemolysins  or  bacterioly- 
sins)  only  when  joined  to  the  complement  or  ferment  group. 
They  are  formed  in  the  serum  by  immunization  with  foreign 
cells,  and  in  turn  combine  with  cells. 


SPECIAL    PROPERTIES    OF    BLOOD    SERUM.  279 

Another  product  of  immunization  with  cells  is  the  agglutinin 
receptor,  while  immunization  with  toxins  leads  to  the  forma- 
tion of  receptors  of  the  First  Order.  Cytotoxins  produced  by 
one  animal  species  A,  brought  into  the  serum  of  another  ani- 
mal species  B,  lead  to  the  formation  of  anticytotoxins  which 
may  be  either  anti  complements  or  anti  amboceptors. 

Toxin  molecules  on  standing  or  by  heating  seem  to  lose 
some  of  their  activity  or  toxic  power,  while  their  power  of 
combining  chemically  with  or  neutralizing  antitoxins  is  not 
diminished.  In  this  condition  they  are  called  by  Ehrlich  to:\;- 
oids,  and  he  explains  the  behavior  by  assuming  that  the  toxins 
have  two  characteristic  groups,  one  of  which,  a  haptophorous 
group,  persists  and  combines  with  the  antitoxin  while  the  other 
is  less  stable  and  may  be  lost;  this  he  calls  the  toxophorous 
group.  By  warming  to  55°-6o°  the  complement  bodies  of 
serum  become  converted  into  inactive  complementoids,  which 
retain  the  haptophorous  group  but  lose  the  zymotoxic  group. 
Amboceptors  may  lose  in  the  same  way  one  of  their  combining 
groups  and  become  aniboccptoids. 

It  would  not  be  proper  in  this  place  to  go  more  fully  into 
the  details  of  the  Ehrlich  theory;  enough  has  been  given  to 
furnish  the  student  an  outline  of  the  most  important  points  in 
the  theory.  It  is  of  course  true  that  much  of  the  present  view 
is  artificial  and  tentative  and,  with  closer  fixation  of  facts,  must 
be  modified.  This  has  been  the  history  of  the  development  of 
all  chemical  theories.  In  its  main  features  the  Ehrlich  doc- 
trine gives  us  a  tangible  picture  of  how  the  serum  may  act 
toward  foreign  bodies.  For  the  ultimate  reasons  for  the  for- 
mation of  immune  side  chains  by  stimulation  we  have  no  more 
explanation  than  we  have  for  many  of  the  manifestations  of 
chemical  affinity.  In  its  outlines  this  theory  of  the  action  of 
immune  serum  appears  wholly  fanciful,  but  in  reality  it  makes 
no  greater  claim  on  the  imagination  than  do  some  of  the  old- 
est accepted  theories  of  general  chemistry. 


CHAPTER    XV. 
TRANSUDATIONS   RELATED   TO   THE   BLOOD. 

THE    LYMPH. 

The  capillary  vessels  convey  the  arterial  blood  with  its  store 
of  nutriment  to  the  various  tissues,  which  by  transudation 
receive  the  required  amount  of  nourishing  matter.  The 
transuded  liquid  is  the  lymph  which  serves  the  double  purpose 
of  nourishing  the  tissues  and  draining  them  also,  since  this 
liquid  not  only  gives  up  large  molecules  of  absorbable  matter, 
but  takes  up  at  the  same  time  various  products  of  metabolism. 
What  is  left  over  after  this  contact  with  the  tissues  collects  in 
the  minute  lymph  capillaries  and  then  into  the  larger  lymphatic 
circulation  proper. 

Composition.  Being  thus  related  to  the  blood  the  lymph 
must  have  a  composition  not  greatly  different  from  that  of 
the  plasma.  The  normal  lymph  is  a  nearly  clear  fluid  with  a 
specific  gravity  somewhat  less  than  that  of  the  serum  as  a  rule. 
It  contains  salts  and  organic  substances  as  does  the  serum  of 
the  blood,  but  is  naturally  poorer  in  protein  elements  since  a 
portion  of  these  has  been  taken  up  to  nourish  the  neighboring 
cells.  Very  few  red  corpuscles  are  present,  but  as  the  forma- 
tion of  leucocytes  or  white  corpuscles  takes  place  in  the  so- 
called  lymphatic  glands  these  form  elements  are  abundant  in 
the  final  flow. 

It  has  been  already  shown  that  potassium  salts  are  common 
to  the  corpuscles  of  the  blood,  while  the  salts  of  sodium  are 
abundant  in  the  plasma.  We  naturally  find  the  same  thing  in 
the  lymph  which  contains,  in  addition  to  proteins,  fat,  sugar, 
cholesterol,  etc.,  inorganic  salts,  having  about  the  following 
composition,  according  to  some  old  analyses  by  Schmidt : 

Sodium  chloride    5.67  per  1000 

Sodium   oxide    1.27 

280 


TRANSUDATIONS    RELATED    TO    BLOOD.  261 

Potassium   oxide    0.16  per  1000. 

Sulphuric  acid,  SO3   0.09 

P2O5  as  combined   with  alkalies    0.02 

Calcium  and  magnesium  phosphates    0.26 

This  result  is  approximately  what  one  would  expect  from  an 
analysis  of  blood  serum  and  is  in  fact  about  what  has  been 
found.  The  two  fluids  have  the  same  osmotic  pressure,  due 
largely  in  both  cases  to  salt  content. 

Function  of  the  Lymph.  The  amount  of  metabolic  sub- 
stances returned  finally  to  the  venous  circulation  through  the 
lymphatics  does  not  appear  to  be  great.  The  chief  product 
of  oxidation,  CO2,  seems  to  be  thrown  back  directly  from  the 
lymph  spaces  to  the  smaller  vessels  leading  to  the  venous  sys- 
tem. The  lymph  spaces  into  which  the  transuded  serum  flows 
have  apparently  two  ways  of  discharge.  The  bulk  of  the 
liquid  with  some  of  the  absorbed  metabolic  products  passes,  as 
intimated,  into  the  gradually  enlarging  lymphatic  system,  but 
certain  other  complexes,  and  among  them  possibly  the  most 
abundant  oxidation  products,  evidently  find  their  way  imme- 
diately into  the  capillary  beginnings  of  the  venous  circulation. 
It  is  not  possible  to  give  exact  figures  as  to  the  relative  amounts 
of  these  products  going  the  two  courses,  but  it  is  accurately 
known  that  the  carbon  dioxide  pressure  in  the  lymph  is  much 
less  than  in  the  venous  circulation,  and  the  urea  appears  also 
to  be  less. 

It  appears  to  be  pretty  well  established  that  the  leucocytes 
are  active  in  hastening  the  destruction  of  complex  products 
of  tissue  waste.  These  cells  seem  to  possess  a  marked  chem- 
ical activity  which  is  manifested  in  a  kind  of  digestion  of  the 
grosser  complexes  separated  in  the  tissue  metabolism.  The 
normal  end  products  of  this  breaking  down  process  are  not 
formed  at  once.  Possibly  the  leucocyte  is  one  of  the  assist- 
ing agents.  It  has  been  therefore  held  by  many  writers  that 
the  formation  of  these  lymph  cells  is  probably  the  most  im- 
portant part  of  the  work  in  the  lymphatic  system. 


282  PHYSIOLOGICAL    CHEMISTRY. 

CHYLE. 

During  the  digestion  of  fatty  foods  the  lymph  absorbed 
from  the  intestinal  walls  contains  numerous  minute  fat  glob- 
ules in  the  form  of  an  emulsion.  This  portion  of  the  lymph  is 
known  as  chyle  and  is  carried  along  by  the  lacteals  and  finally 
discharged  into  the  lower  part  of  the  thoracic  duct. 

In  composition  chyle  differs  from  the  lymph  from  other 
sources  mainly  in  its  fat  content.  In  the  periods  when  diges- 
tion is  not  in  progress  the  lacteal  lymph  is  also  clear. 

TRANSUDATIONS  PROPER. 

The  lymph  has  sometimes  been  considered  a  transudation 
of  the  blood,  but  the  term  is  now  more  commonly  used  to 
describe  the  flow  of  liquid  from  the  blood  into  certain  cavities 
of  the  body  under  pathological  conditions.  x\  transudation 
proper  is  then  a  modified  lymph  and  results  often  from  an 
imperfect  elimination  of  water  by  the  kidneys,  or  from  some 
disturbance  in  the  circulation.  Inflammatory  transudations 
are  sometimes  distinguished  as  exudations. 

For  example,  the  pleural  and  peritoneal  cavities  contain  but 
little  fluid.  The  serous  surfaces  are  moist,  but  it  would  not  be 
possible  to  collect  enough  fluid  substances  for  satisfactory  an- 
alysis under  normal  conditions.  In  the  advanced  stage  of 
pleurisy  a  considerable  quantity  of  fluid  collects  in  the  pleural 
cavity  and  its  composition  resembles  that  of  the  lymph,  but  it 
is  poorer  in  solids  ordinarily.  In  some  forms  of  acute  peri- 
tonitis a  collection  of  similar  fluid  may  take  place  in  the  peri- 
toneal cavity  and  this  may  amount  in  bad  cases  to  se\eral 
liters. 

The  various  forms  of  dropsy  described  by  physicians  are 
essentially  characterized  by  analogous  transudations  of  serous 
fluid  without  inflammation.  Ascites,  or  dropsy  of  the  ab- 
domen, hydrocele,  or  dropsy  of  the  testicle,  and  hydrothorax, 
dropsy  of  the  pleura,  are  illustrations.  Some  analyses  are 
given  below,  showing  the  general  nature  of  the  fluids  collected 


TRANSUDATIONS    RELATED    TO    BLOOD.  283 

in  such  cases.  It  must  not  be  supposed,  however,  that  exactly 
similar  results  would  always  be  obtained  by  analyses  of  fluids 
from  the  same  organs.  The  composition  of  pus  serum  is 
somewhat  similar;  it  contains,  however,  more  products  of 
protein  disintegration. 

Hydrocele  „      c- 

Fluid  ,u^"'l"T, 

(Hammarsten).  (Hoppe-Seyler). 

Water    938.85        Water    909.63 

Serum   albumin    35-94        Proteins    70.22 

Globulin    1325         Lecithin     1.03 

Fibrin    0.59         Fat    0.27 

Ether   extractives    4.02        Cholesterol   0.70 

Soluble  salts   8.60  Alcohol  extractives  ....  1.13 

Insoluble   salts    0.66        Water  extractives   9.22 

Salts    7.75 

Pleural  Peritoneal 

Transudate  Transudate 

(Scherer).  (Hoppe-Seyler). 

Water    935-52        Water    969.64 

Albumin    49-77        Albumin    19-29 

Fibrin    0.62        Urea 0.31 

Ether  extract    2.14         Ether  extract    0.43 

Alcohol  extract    1.84        Alcohol  extract    1.37 

Water   extract    1.62        Water   extract    0.98 

Salts     7.93        Salts  7.98 

Amniotic  Fluid.  This  may  be  considered  as  a  kind  of 
transudate.  A  number  of  analyses  have  been  made  which 
show  about  98.5  per  cent  of  water,  i  per  cent  of  salts  and  0.5 
per  cent  of  organic  solids,  largely  proteins. 

THE  LYMPH  CELLS. 

These  large  cells  or  leucocytes  have  already  been  referred 
to  as  formed  in  the  lymph  glands.  They  are  also  formed  in 
large  numbers  in  the  spleen  and  the  thymus  gland.  From 
whatever  source  produced  they  are  supposed  to  have  the  same 
general  composition  and  chemical  function.  The  following 
analysis  by  Lilienfeld  gives  an  idea  of  their  general  composi- 
tion. The  dry  substance  of  the  cells  amounted  to  114.9  parts 
per  1000  and  was  made  up  of  the  following  constituents,  the 


284  PHYSIOLOGICAL    CHEMISTRY. 

figures  referring  to  per  cent  amounts  of  the  dry  matter.     Cells 
from  thymus  of  calf. 

Leuco-nuclein    68.79 

Albumins    1.76 

Histone 8.67 

Lecithin    7-51 

Fat   402 

Cholesterol    4-40 

Glycogen     0.80 

In  addition  to  these  organic  substances  mineral  matters  are 
present,  with  salts  of  potassium  characteristic. 

Pus  Cells.  In  their  origin  and  characteristics  these  may 
be  considered  as  very  similar  to  the  leucocytes,  if  not  indeed 
identical  with  them.  The  few  analyses  made  show  a  general 
agreement  when  reduced  to  the  same  terms.  It  must  be  re- 
membered that  such  analyses  are  far  from  simple  operations, 
especially  in  the  separation  of  the  several  protein  constituents. 
The  following  figures  by  Hoppe-Seyler  should  be  compared  in 
that  light  with  the  above.  The  numbers  refer  as  before  to  the 
dr\'  matter : 

Nuclein   and   albumin    67.40 

Lecithin    7-56 

Fat   750 

Cholesterol    728 

Cerebrin  and  extractives    10.28 

The  Spleen.  While  our  knowledge  of  the  functions  of  the 
spleen  is  very  imperfect  a  few  words  may  be  said  in  this  con- 
nection, since  as  far  as  is  known  the  lymph  glands  in  produc- 
ing leucocytes  do  about  the  same  kind  of  work.  The  one 
thing  most  apparent  about  the  spleen  is  that  in  this  organ  large 
numbers  of  white  cells  are  formed  and  given,  to  the  blood ;  it 
is  also  known  that  these  cells  suffer  destruction  there,  as  the 
spleen  pulp  contains  considerable  quantities  of  the  xanthine 
bases,  which  are  among  the  common  products  of  cell  nuclei 
destruction.  The  cells  so  destroyed  may  possibly  be  those 
which  have  already  served  their  purpose  in  the  blood  as  the 


TRANSUDATIONS    RELATED    TO    BLOOD.  285 

disintegrating  agents  concerned  in  the  breaking  down  of  other 
bodies.  Uric  acid,  as  derived  from  the  xanthine  bases,  is 
known  to  resuh  when  blood  is  rubbed  up  with  the  spleen  sub- 
stance. The  spleen  is  enlarged  in  many  cases  of  infectious 
diseases.  This  is  possibly  from  the  abnormally  great  produc- 
tion of  leucocytes  needed  in  the  blood  in  overcoming  the  effects 
of  the  toxic  agents  or  invading  bacteria. 

Of  the  chemical  nature  of  the  spleen  substance  little  is 
known,  as  it  is  practically  impossible  to  free  it  from  blood  for 
analysis.  In  addition  to  the  xanthine  bodies  and  other  decom- 
position products  there  seems  to  be  present  an  albuminous 
substance  containing  iron,  which  is  considered  as  an  albu- 
minate; but  of  its  uses  nothing  definite  is  known. 


CHAPTER    XVI. 

MILK. 

The  qualitative  composition  of  milk  as  produced  by  the 
mammary  glands  of  different  animals  is  nearly  the  same  what- 
ever the  species  of  animal.  But  in  quantitative  composition 
very  great  differences  obtain.  Cow's  milk  has  always  been 
taken  as  the  type  with  which  comparisons  are  made,  as  it  is 
the  kind  everywhere  in  general  use.  The  essential  differences 
between  it  and  mother's  milk  will  be  pointed  out  in  what 
follows. 

COW'S  MILK. 

In  an  earlier  chapter  an  analysis  of  cow's  milk  is  given 
which  represents  a  general  average  of  composition  of  good 
market  milk.  But  the  normal  milk  of  individual  cows  may 
be  very  different  from  that  there  described.  The  qualitative 
composition  is  always  the  same,  but  in  the  amounts  of  fat, 
sugar  and  protein  present  the  greatest  divergences  are  noticed. 
These  variations  depend  on  the  race  of  the  animal,  the  period 
of  lactation  and  especially  on  the  feed.  It  is  also  a  well- 
known  fact  that  the  richness  of  milk  varies  during  the  time 
of  milking,  the  first  portions  of  milk  withdrawn  from  the 
udder  being  poorer  in  fat  than  the  last  part  or  "  strippings." 
In  speaking  of  normal  milk,  then,  these  facts  must  be  kept  in 
mind ;  a  milk  may  be  normal  but  not  necessarily  rich  or  good, 
from  the  standpoint  of  food  value.  The  following  table  illus- 
trates the  variations  found  in  the  analyses  of  milk  from  a  large 
number  of  cows.  The  mean  specific  gravity  is  from  1.029  to 
I-033- 

Mean.  Maximum.  Minimum. 

Water    87.4  91.5  84.0 

Fat  3.5  6.2  2.0 

Sugar    4.5  6.1  2.0 

Proteins    3.9  6.6  2.0 

Salts    0.7  i.o  0.3 

r86 


MILK.  287 

When  the  water  of  milk  is  found  as  high  as  91.5  per  cent 
of  the  whole  the  sum  of  the  fat,  protein,  sugar  and  salts  can 
be  only  8.5  per  cent  in  place  of  12.5  per  cent,  which  should  be 
expected  in  the  mixed  market  milk. 

Market  Milk.  Experience  has  shown  that  the  mixed  milk 
from  a  herd  of  well-kept  cows  should  have  a  composition  not 
far  from  that  given  in  the  table  above  under  "  mean."  Laws 
have  been  passed  in  most  of  the  large  cities  of  the  United 
States  and  Europe  requiring  that  milk  sold  as  pure  must  be 
of  a  quality  not  inferior  to  this  mean  value.  Indeed,  in  some 
places  a  market  milk  of  still  higher  standard  is  required. 

PHYSICAL  COMPOSITION   OF   MILK. 

The  exact  nature  of  the  mixture  of  the  component  parts  of 
milk  has  long  been  a  debated  question.  When  taken  from 
the  udder  the  fats,  proteins,  sugar  and  salts  are  mixed  homo- 
geneousl}',  and  no  immediate  tendency  is  observed  toward  a 
separation  of  the  light  fat  from  the  other  and  heavier  solids. 
In  time,  however,  such  a  separation  takes  place  and  the  fat 
rises  in  the  form  of  cream.  Milk  cannot  be  looked  upon  as 
a  transudation  from  the  blood  because  it  contains  substances 
not  found  in  that  fluid;  the  casein  of  milk  and  the  lactose  are 
different  from  the  proteins  and  sugar  normally  existent  in 
the  blood,  and  the  fat  of  milk  is  more  complex  probably  than 
the  blood  fat.  It  is  necessary  to  admit,  then,  that  some  of 
the  milk  components  are  produced  in  or  from  the  substance  of 
the  mammary  glands  themselves.  It  is  held  by  some  authori- 
ties that  the  nucleo-proteids  of  the  gland  cells  are  similar  to 
or  identical  with  the  casein,  which  therefore  has  its  origin  in 
the  gradual  breaking  down  of  those  cells.  In  regard  to  the 
milk  fat  it  is  known  that  certain  fats  can  pass  but  little  changed 
from  the  food  through  the  blood  and  appear  finally  in  the 
milk,  imparting  peculiar  properties.  But,  on  the  other  hand, 
milk  fat  is  produced  when  the  food  of  the  animal  contains  no 
fat  whatever,  and  certainly  no  fats  resembling  the  characteris- 
tic volatile  fats  of  the  milk.     In  the  carnivora,  confined  to  an 


288 


PHYSIOLOGICAL    CHEMISTRY 


essentially  protein  diet,  milk  fat  is  formed,  and  in  the  herbi- 
vora  on  a  diet  containing-  largely  pentoses  and  other  carbo- 
hydrates milk  fat  is  likewise  produced  normally  and  in 
quantity. 

These  facts  then  seem  to  be  clear,  that  while  under  some 
circumstances  fats  as  such  pass  from  the  blood  into  the  milk, 
and  this  is  further  evident  by  the  experience  of  feeding  cows 
with  certain  foods  rich  in  fats,  the  milk  glands  have  the 
power  of  producing  the  several  individual  fats  as  occurring  in 
milk  from  compounds  which  are  not  fat  to  begin  with.  In 
discussing  the  chemistry  of  proteins  in  an  earlier  chapter  it 
was  shown  that  in  the  breaking  down  of  these  bodies  under 
the  influence  of  various  agents  fatty  acids  are  found  among 
the  decomposition  products.  The  complex  protein  molecule 
may  be  all  that  is  necessary  to  give  rise  to  the  milk  fats  if 
other  things  are  not  available. 

The  origin  of  milk  sugar  is  not  at  all  clear.  Lactose  is  not 
a  constituent  of  our  ordinary  foods  and  at  best  the  blood  con- 
tains probably  only  inverted  sugars  or  monosaccharides.  In 
some  cases  the  formation  of  milk  sugar  may  be  traced  indi- 
rectly to  the  carbohydrates  of  the  food ;  but  this  will  not  ex- 
plain the  production  of  sugar  in  the  carnivora.  Here  as  be- 
fore we  are  probably  obliged  to  fall  back  on  the  behavior  of 
the  complex  proteins.  Among  the  groups  they  contain,  or  at 
any  rate  yield  in  decomposition,  the  presence  of  sugar  groups 
has  been  certainly  shown.  This  was  explained  in  a  former 
chapter.  The  milk  lactose  probably  results  from  a  synthesis 
of  these  simpler  sugars. 

From  what  is  in  general  known  of  the  nature  of  complex 
protein  matter  such  as  exists  in  the  milk  glands  it  seems  there- 
fore possible  to  trace  the  origin  of  the  milk  proteins,  sugar  and 
fats  to  the  disintegration  of  this  original  protein  substance. 
But  of  the  agents  of  disintegration,  and  following  necessary 
syntheses,  we  know  absolutely  nothing.  The  presence  of  cer- 
tain enzymes  has  been  assumed,  but  as  they  have  not  been 
isolated  or  identified,  their  part  in  the  reactions  remains 
speculative. 


MILK.  289 

CHEMISTRY  OF  THE  MILK  COMPONENTS. 

Fats.  In  the  older  literature  milk  fat  was  given  a  com- 
paratively simple  composition.  It  was  assumed  to  consist  of 
stearin,  palmitin,  olein  and  butyrin  essentially,  the  last  named 
volatile  fat  imparting  the  flavor  to  the  separated  butter.  At 
the  present  time  we  must  admit  that  our  knowledge  is  far  from 
exact  on  the  subject,  but  we  know  that  the  composition  of  milk 
fat  is  by  no  means  as  simple  as  once  assumed.  In  the  chapter 
on  the  fats  an  analysis  is  given  which  conforms  better  to  our 
modern  notions.  We  find  then  besides  butyrin  several  gly- 
cerol esters  of  the  same  series  of  comparatively  volatile  acids. 
Among  the  heavier  fatty  acids  myristic  acid  seems  to  have 
some  importance,  as  disclosed  by  a  number  of  analyses.  A 
small  amount  of  lecithin  appears  to  be  also  present. 

Although  produced  from  a  variety  of  materials  in  feeding 
experiments,  milk  fat,  as  butter,  maintains  a  rather  constant 
composition  as  disclosed  by  both  chemical  and  physical  tests. 
The  melting  point  of  the  fat  is  usually  between  31°  and  32.5° 
C.  and  its  specific  gravity  at  38°  is  about  0.912.  Butter  fat 
is  easily  saponified  and  from  the  saponified  mass  the  fatty 
acids  which  are  non-volatile  and  practically  insoluble  in  hot 
water  may  be  separated.  These  insoluble  acids  amount  in  the 
mean  to  about  87.5  per  cent  of  the  weight  of  the  original  but- 
ter fat.  That  is  10  grams  of  average  butter  fat  should  yield 
8.75  gm.  of  insoluble  acids,  the  difference  representing  the 
lighter  soluble  fatty  acids  and  glycerol.  In  the  fat  from  very 
rich  milk  the  insoluble  acids  may  be  somewhat  lower  than  this, 
while  in  poor  milk  they  would  be  higher.  These  facts  are 
important  in  distinguishing  between  butter  and  its  substitutes. 

Fat  Globules.  In  milk  the  fat  exists  in  the  form  of  minute 
globules  of  different  sizes.  The  diameters  of  these  globules 
vary  between  about  0.0016  mm.  and  0.0 1  mm.  A  cubic  centi- 
meter of  normal  milk  containing  3.5  per  cent  of  fat  contains 
100  millions  or  more  of  these  globules.  In  the  milk  they  are 
described  as  existing  in  the  form  of  an  emulsion,  but  of  the 


290  PHYSIOLOGICAL    CHEMISTRY. 

exact  nature  of  this  emulsion  our  knowledge  is  imperfect.  It 
has  been  held  also  that  a  membrane  of  casein  encloses  the  fat 
globules  and  that  this  prevents  the  ready  extraction  of  fat 
when  ether  or  similar  solvent  has  been  added  to  milk.  If  the 
milk  is  previously  shaken  with  a  little  acid  which  is  supposed 
to  break  or  destroy  this  membrane  the  ether  added  will  now 
dissolve  it.  But  this  membrane  cannot  be  directly  detected 
with  the  microscope  and  experiments  on  the  formation  of  fat 
emulsions  by  the  aid  of  casein  and  weak  alkalies  have  shown 
that  the  presence  of  a  membrane  is  not  necessary  to  account 
for  the  round  form  or  the  failure  to  dissolve  readily  in  ether. 
The  surface  of  the  globule  is  not  the  same  as  the  interior  por- 
tion, as  it  appears  to  take  a  stain  by  certain  agents  which  does 
not  penetrate.  But  in  the  conflict  of  views  advanced  it  is  not 
yet  known  what  the  surface  actually  is. 

Casein  and  Lactalbumin.  These  compounds  have  been 
mentioned  in  the  chapter  on  proteins  and  their  place  in  the 
general  scheme  of  classification  pointed  out.  In  the  free,  pure 
state  the  casein  is  a  distinctly  acid  body  which  neutralizes  al- 
kali and  forms  salts  with  rather  sharply  defined  properties. 
Casein  may  be  easily  separated  from  milk  in  this  way : 

Ex.  Dilute  500  cc.  of  skimmed  milk  with  about  2  liters  of  water  in  a 
large  jar;  add  enough  dilute  acetic  acid  to  make  not  over  o.i  per  cent 
of  the  whole.  This  causes  a  precipitation  of  the  casein  in  fine  white 
flakes  which  soon  settle,  leaving  a  nearly  clear  whey.  After  some  hours 
decant  this  whey  and  add  a  greater  volume  of  distilled  water  and  stir  up 
well.  Allow  this  mixture  to  settle  and  pour  off  the  water.  Add  a  liter  of 
water  and  enough  weak  sodium  or  ammonium  hydroxide  to  dissolve  all 
the  casein  and  produce  an  opalescent  solution.  This  in  turn  is  re- 
precipitated  with  dilute  acetic  acid  after  adding  considerable  water,  and 
these  operations  are  repeated  several  times.  In  this  way  a  casein  nearly 
free  from  calcium  salts  is  obtained.  It  is  washed  well  with  water  by 
decantation,  then  poured  on  a  Buchner  funnel,  drained,  washed  with  alco- 
hol, until  the  water  is  removed  and  finally  several  times  with  ether  to 
take  out  the  fat.  On  drying  a  fine  white  powder  is  obtained  with  which 
the  important  properties  of  casein  may  be  shown. 

Ex.  Weigh  out  5  to  10  gms.  of  casein  into  a  beaker  or  fiask  and  add 
distilled  water.  Note  that  it  appears  to  be  quite  insoluble.  (This  might 
be  shown  by  filtering  and  testing  the  filtrate  by  evaporation.)  Add  a  few 
drops  of  phenol-phthalein  reagent  and  run  in  standard  sodium  hydroxide 


MILK.  291 

solution  until  a  permanent  pink  appears.  In  this  way  the  equivalent  or 
combining  weight  of  the  casein  may  be  found  very  closely.  It  is  over 
1000. 

The  alkali  salt  of  the  casein  forms  a  somewhat  viscid  solution.  If  ex- 
posed to  the  air  it  dries  down  to  a  gummy  mass  which  is  very  adhesive 
and  acts  like  a  mucilage.  In  the  arts  similar,  but  crude,  solutions  are 
used  as  sizing  material  and  as  a  constituent  of  certain  paints.  These 
products  are  made  from   the  cheap  whey  from  the  creameries. 

Ex.  While  the  alkali  salts  of  casein  are  readily  soluble  in  water  the 
heavier  metal  combinations  are  not.  This  may  be  shown  by  adding  to 
the  alkali  solution  as  obtained  above  solutions  of  salts  of  other  metals. 
Precipitates  are  formed  readily  with  most  of  them.  The  calcium  salt  is 
moderately  soluble,  as  may  be  shown  by  rubbing  up  some  casein  with 
calcium  carbonate  and  water.  On  filtering  and  adding  a  drop  of  acetic 
acid  to  the  filtrate  a  casein  precipitate  comes  down. 

Ex.  The  lactalbnmin  may  be  shown  by  boiling  the  decanted  liquid  from 
the  first  acetic  acid  precipitation  given  above.  A  coagulum  forms  as  in 
a  dilute  white  of  egg  solution  to  which  a  little  acid  had  been  added. 

The  phosphorus  in  casein  appears  to  be  combined  in  at  least 
two  forms.  On  digesting  casein  with  pepsin  and  hydrochloric 
acid  a  product  known  as  pseudo-nuclein  is  separated  because 
of  its  failure  to  digest.  In  long  continued  digestion  some 
phosphorus  seems  to  pass  into  the  form  of  orthophosphoric 
acid,  while  another  portion  remains  in  the  albumoses  formed, 
in  the  organic  condition.  The  digestion  residue,  however,  is 
not  nucleinic  acid,  which  distinguishes  the  casein  from  the  true 
nucleo-proteids.  In  the  precipitation  of  casein  from  milk  by 
the  treatment  given  above  the  combined  phosphorus,  whether 
in  acid  or  organic  combination,  does  not  appear  to  be  touched. 
The  mineral  phosphates  are  separated,  however,  but  not  com- 
pletely, as  the  finally  washed  and  dried  casein  always  contains 
some  ash,  a  part  of  which  is  calcium  phosphate. 

Milk  Sugar.  This  crystallizes  with  one  molecule  of  water, 
C12H22O11  +  H2O,  and  yields  glucose  and  galactose  on  inver- 
sion. It  is  separated  in  large  quantities  from  the  whey  of 
cheese  factories  and  is  employed  in  the  manufacture  of  invalid 
and  infant  foods.  The  general  properties  of  the  sugar  have 
already  been  given. 

The  Mineral  Substances  in  Milk.  In  the  analyses  quoted 
at  the  beginning  of  the  chapter  the  ash  of  the  milk  is  given  as 


292  PHYSIOLOGICAL    CHEMISTRY, 

about  0.7  per  cent  in  the  mean.  This  amount  appears  small, 
but  still  it  is  of  the  highest  importance,  as  it  makes  up  between 
5  and  6  per  cent  of  the  total  solids  of  the  milk.  The  composi- 
tion of  milk  ash  has  been  the  subject  of  many  investigations. 
While  it  cannot  represent  exactly  the  condition  of  the  inor- 
ganic substances  in  the  original  milk,  the  agreement  is  an  ap- 
proximate one  and  is  probably  near  enough  for  practical  pur- 
poses. In  obtaining  ash  for  analysis,  sulphur  and  phosphorus 
in  organic  combination  are  thrown  into  oxidized  form  and 
combined  as  salts,  sulphates  and  phosphates,  in  which  form 
we  find  them  in  our  subsequent  tests.  The  following  figures 
from  Konig  represent  the  composition  of  milk  ash  as  the 
mean  of  9  analyses : 

IVr  L  ent. 

K:0     24.06 

NajO    6.05 

CaO    23.17 

:MgO     2.63 

FejOa  0.44 

P=05    27.98 

SO,     1.26 

CI     1345 

Accepting  these  figures  as  fairly  accurate,  and  they  agree 
pretty  well  with  the  results  of  all  analysts  who  have  dealt  with 
the  question,  i  liter  of  average  cow's  milk  would  contain  the 
following  amounts  of  the  several  constituents : 

K.0   1.74  gm. 

NasO    0.44  gm. 

CaO    1.67  gm. 

MgO    o.  19   gm. 

FciOs     0.03    gni. 

P2O5     2.02    gm. 

SOj    0.09    gm. 

CI    0.97  gm. 

715 

Noteworthy  here  are  the  relatively  large  amounts  of  the 
phosphates  of  calcium  and  potassium.  These  salts  represent 
all  the  mineral  matters  needed  in  nourishing  the  body.     As 


MILK.  293 

found  in  the  milk  they  exist  in  the  combinations  from  which 
they  are  most  readily  assimilated. 

The  Colostrum.  This  is  the  milk  secreted  before  and  for 
a  few  days  after  parturition,  and  is  characterized  by  higher 
specific  gravity  and  content  of  solids.  It  contains  a  large 
amount  of  coagulable  proteins  and  therefore  thickens  on  boil- 
ing. Some  idea  of  the  general  composition  is  given  by  the 
following  figures  which  represent  the  means  of  a  number  of 
analyses : 

Per  Cent 

Water     74.05 

Casein     4.66 

Albumin      1362 

Fat     3-43 

Sugar      2.66 

Salts    1.58 

Whey  is  the  fluid  left  after  separation  of  the  larger  part  of  the  fat  and 
casein  in  the  cheese  industry  or  by  analogous  coagulation.  The  sugar  and 
salts  remain  practically  unchanged,  while  the  fat  and  casein  are  reduced  to 
traces.      The  lactalbumin  left  averages  about  0.5  per  cent. 

Buttermilk  differs  from  ordinary  milk  essentially  in  its  lower  content 
of  fat.  It  is  usually  sour  because  of  being  separated  from  ripened  cream, 
and  contains  therefore  an  appreciable  amount  of  lactic  acid  formed  at  the 
expense  of  some  of  the  sugar. 

Skimmed  milk  is  in  composition  similar  to  buttermilk  but  is  usually 
sweet.  In  the  modern  methods  of  separation  by  centrifugal  machines  the 
fat  may  be  reduced  to  less  than  half  of  one  per  cent;  the  protein  is  also 
somewhat  reduced. 

SOME    EXPERIMENTS    WITH    MILK. 

A  few  simple  tests  may  be  made  to  illustrate  the  composi- 
tion of  milk. 

The  Test  for  Fat.  Pour  about  20  cc.  of  milk  in  a  porcelain  dish,  add 
an  equal  volume  of  clean,  dry  quartz  sand  and  evaporate,  with  frequent 
stirring,  about  an  hour  on  the  water-bath.  Then  loosen  the  dry  mass  as 
well  as  possible  by  means  of  a  spatula,  or  glass  rod,  and  pour  over  it  25  cc. 
of  light  benzine.  Stir  up  well  and  cover  with  a  sheet  of  paper  and  allow 
to  stand  15  minutes.  Then  pour  the  liquid  through  a  small,  dry  filter  into 
a  small,  dry  beaker,  and  place  this  in  hot  water  to  volatilize  the  benzine. 

A  residue  of  fat  will  be  left.  Do  not  attempt  to  evaporate  the  benzine 
over  a  flame,  or  on  a  water-bath  under  which  a  lamp  is  burning.  Heat  the 
water,  then  extinguish  the  flame  and  immerse  the  vessel  containing  the 
benzine  in  the  hot  water. 


294  PHYSIOLOGICAL    CHEMISTRY. 

The  Test  for  Sugar.  Measure  out  about  lo  cc.  of  milk,  and  dilute  it 
with  water  to  make  200  cc.  Add  to  this  5  cc.  of  a  copper  sulphate  solu- 
tion, such  as  is  used  in  making  the  Fehling  solution  (69.3  gm.  per  liter), 
and  then  enough  potassium  or  sodium  hydroxide  solution  to  produce  a 
voluminous  precipitate  containing  copper  with  all  the  proteins  and  fat. 
For  this  purpose  about  3.5  cc.  of  a  i  per  cent  sodium  hydroxide  solution 
will  be  required.  Allow  the  precipitate  to  subside,  pour  or  filter  off  some 
of  the  supernatant  liquid,  and  boil  it  with  Fehling  solution.  The  character- 
istic red  precipitate  forms,  showing  presence  of  sugar. 

Protein  Test.  The  presence  of  proteins  in  milk  can  readily  be  shown 
as  follows :  Mix  equal  volumes  of  milk  and  Millon's  reagent  in  a  test-tube, 
and  boil.  The  bulky  red  precipitate  which  forms  proves  the  presence  of 
the  body  in  question. 

Action  of  Rennet  on  Milk.  The  mucous  membrane  of  the  stomachs  of 
most  animals,  and  especially  that  of  the  young  calf,  contains  an  enzyme 
known  as  the  "milk  curdling  ferment,"  the  "rennet  ferment,"  or  rennin, 
the  nature  of  which  has  already  been  explained  in  the  chapter  on  the  fer- 
ments. 

A  crude  extract  of  the  mucous  membrane  of  the  stomach  from  the  calf 
is  commonly  called  rennet  and  has  long  been  in  use  for  the  curdling  of 
milk  in  the  production  of  cheese.  This  curdling  consists  essentially  in  the 
coagulation  or  precipitation  of  the  casein,  which,  it  will  be  recalled,  is 
not  readily  thrown  down  b\'  the  usual  methods. 

An  active  rennet  can  be  readily  obtained  by  digesting  the  stomach  of  the 
calf  with  glycerol  or  brine.  If  a  brine  extract  is  precipitated  by  alcohol 
in  excess  a  white  powder  separates,  which,  when  collected  and  dried,  has 
very  active  properties.  Several  powders  of  this  description  are  now  in  the 
market.    Let  the  student  try  the  following  experiment  with  such  a  product : 

Ex.  Warm  some  fresh  milk  to  a  temperature  of  38°  to  40°  C.  in  a  test- 
tube  or  small  beaker,  then  add  about  half  a  gram  of  commercial  "  rennin," 
and  after  stirring  it  well  keep  for  15  minutes  at  a  temperature  not  above 
40°.  Then  as  the  milk  cools  it  assumes  the  consistence  of  a  firm  jelly.  It 
is  essential  in  this  experiment  that  the  temperature  be  kept  within  the 
proper  limits,  as  the  enzjme  is  not  active  at  low  temperature  and  it  is, 
like  others,  destroyed  by  high  temperature.  The  casein  or  cheese  which  is 
obtained  in  this  way  is  not  the  same  as  that  precipitated  by  acids  as  it 
contains  much  calcium  in  combination.  This  form  of  casein  is  usually 
called  para-casein. 

Repeat  the  experiment  by  adding  about  5  drops  of  a  concentrated  sodium 
carbonate  solution  to  the  milk  and  then  the  rennet.  Coagulation  now 
fails  or  is  partial. 

The  Action  of  Pancreatic  Extract  on  Milk.  The  behavior  of  milk  with 
extract  of  pancreas  is  somewhat  complicated  because  of  the  complex 
nature  of  the  milk;  the  sugar,  the  fat,  and  the  protein  bodies  all  suffer 
some  change  under  the  influence  of  the  several  pancreatic  enzymes. 

The  most  interesting  of  these  changes,  however,  is  that  produced  in  the 
proteins,  and  is  commonly  called  peptonization. 


MILK.  295 

At  the  present  time  the  digestion,  or  peptonization  of  milk,  is  a  very- 
common  practice  in  the  preparation  of  food  for  the  sick  room,  and  can  be 
illustrated  by  the  following  experiment : 

Ex.  Dilute  about  10  cc.  of  milk  with  an  equal  volume  of  water,  and  add 
half  a  gram  of  sodium  bicarbonate.  Next  add  a  few  drops  of  a  liquid  ex- 
tract of  pancreas,  or  a  very  small  amount  (10  to  20  mg.)  of  one  of  the 
concentrated  "pancreatin"  powders  on  the  market.  Shake  the  mixture 
and  keep  it  at  a  temperature  of  40  degrees  on  the  water-bath  half  an  hour. 
At  the  end  of  this  time  filter  and  apply  the  peptone  test — ^potassium  hy- 
droxide and  dilute  copper  sulphate — and  observe  the  pink  color.  As  the 
action  of  the  pancreatic  extract  is  continued  the  liquid  resulting  becomes 
very  bitter  from  the  formation  of  digestion  products  other  than  "peptone." 
The  reaction  should  therefore  be  checked  by  cooling  before  this  very  bitter 
stage  is  reached. 

It  will  be  observed  that  these  experiments  illustrate  the  con- 
ditions in  two  kinds  of  digestion.  The  pancreatic  digestion 
of  proteins  in  milk  is  favored  by  a  neutral  or  slightly  alkaline 
reaction.  Alkali  interferes  with  the  rennet  coagulation.  In 
the  stomach  the  clotting  of  the  milk  is  favored  by  the  combined 
action  of  the  acid  and  ferment.  In  the  normal  stomach  coag- 
ulation the  presence  of  calcium  salts  seems  to  be  essential.  If 
milk  be  treated  with  a  small  amount  of  sodium  oxalate  solution 
and  then  rennet,  coagulation  fails.  Calcium  chloride  solution 
added  later,  the  proper  temperature  being  meanwhile  main- 
tained, brings  it  about. 

THE  ANALYSIS   OF  MILK. 

The  above  experiments  suggest  some  of  the  steps  in  the 
quantitative  analysis  of  milk,  a  brief  outline  of  which  follows : 

Water  and  Total  Solids.  Weigh  out  about  5  grams  in  a  small  platinum 
dish  and  evaporate  to  dryness  over  a  water-bath  which  requires  some 
hours.  Then  transfer  the  dish  to  a  hot  air  oven  and  maintain  at  a 
temperature  of  105°  through  half  an  hour.  Cool  the  dish  in  a  desiccator 
and  weigh.  The  loss  of  weight  represents  the  water,  as  practically  nothing 
else  of  consequence  is  volatile. 

Ash  or  Mineral  Matter.  After  weighing  the  dry  residue  or  total  solids 
above  place  the  dish  on  a  triangle  over  a  clear  Bunsen  flame  and  heat 
until  all  the  organic  matter  and  finally  the  excess  of  carbon  is  driven  off. 
The  ash  left  must  be  perfectly  white.  Cool  and  weigh  as  before.  There 
is  some  slight  loss  of  volatile  salts  in  this  ignition. 

Fat.     Where  many  analyses  are  made  as  a  routine  operation,  as  in  the 


296  PHYSIOLOGICAL    CHEMISTRY. 

control  of  market  milk,  fat  is  generally  now  determined,  by  separating  it 
from  the  milk  in  a  centrifugal  machine  and  reading  oflf  the  volume.  A 
definite  quantity  of  milk  is  measured  out,  mixed  with  a  little  acid  to  facili- 
tate the  breaking  up  of  the  fat  globules,  placed  in  a  special  bottle  with 
graduated  neck  or  stem  and  rapidly  rotated.  The  liberated  fat  collects  in 
the  stem  and  is  read  off.  With  the  Babcock  machine  in  common  use  the 
method  is  rapid  and  very  accurate. 

Fat  is  very  frequently  determined  by  evaporating  milk  mixed  with 
broken  glass  or  quartz  sand  to  dryness  and  extracting  with  a  good  sol- 
vent, preferably  light  petroleum  benzine  or  perfectly  anhydrous  ether.  A 
better  method  is  to  distribute  about  5  to  10  grams  of  milk  from  a  pipette 
over  the  surface  of  a  strip  of  specially  prepared  absorbent  paper.  This 
is  coiled  up  somewhat  loosely,  placed  in  an  air  oven,  dried  thoroughly  and 
then  transferred  to  a  Soxhlet  extraction  apparatus,  where  it  is  treated 
with  the  solvent  by  percolation  through  two  or  three  hours.  The  solvent 
carries  the  fat  down  into  a  small  weighed  flask.  On  evaporation  of  the 
solvent  the  dry  fat  is  left  and  may  be  so  weighed. 

Sugar,  To  determine  the  sugar  the  fat  and  proteins  must  be  first  sepa- 
rated, which  may  be  conveniently  done  by  the  copper  process  as  illustrated 
above.  25  cc.  of  milk  is  diluted  with  water  to  400  cc.  and  10  cc.  of  the 
Fehling  copper  solution  added.  Then  from  a  corresponding  sodium 
hydroxide  solution  (containing  10.2  gm.  to  the  liter)  alkali  is  added  in 
amount  just  sufficient  to  throw  down  a  bulky  precipitate  containing  all 
the  proteins  and  fats  with  the  copper.  This  requires  about  7  cc.  of  the 
alkali.  The  mixture  is  diluted  to  500  cc.  and  a  portion  is  filtered  off  for 
tests.  If  the  precipitation  was  properly  made  a  clear  filtrate  is  secured 
which  contains  only  a  trace  of  copper,  and  not  enough  to  appreciably  affect 
the  accuracy  of  titration  by  the  Fehling  solution  as  described  in  an  earlier 
chapter.     The  proper  factor  for  lactose  must  be  used  in  the  calculation. 

The  protein  and  fat  may  be  precipitated  by  use  of  a  solution  of  lead 
acetate  or  mercuric  nitrate  without  dilution.  On  filtering  a  clear  filtrate 
is  obtained  which  may  be  tested  by  the  polariscope.  50  cc.  of  milk  should 
be  used,  and  after  precipitation  and  filtration  made  up  to  100  cc.  for  the 
polarization  test.     The  details  cannot  be  given  here. 

The  Proteins.  If  the  sugar,  fat  and  ash  are  accurately  found  the  pro- 
teins may  be  estimated  by  difference ;  that  is  by  subtracting  the  sum  of 
these  from  the  total  solids  found.  But  this  plan  should  not  be  followed 
except  as  a  control.  A  direct  determination  of  casein  may  be  made  in 
this  way :  10  cc.  of  milk  is  diluted  to  50  and  mixed  with  dilute  acetic 
acid  to  produce  complete  precipitation.  Something  less  than  1.5  cc.  of  10 
per  cent  acid  will  be  needed  for  this.  The  precipitate  is  collected  on  a 
Gooch  funnel,  washed  with  water,  hot  alcohol  and  finally  enough  ether 
to  remove  all  the  fat.  What  is  left  is  dried  and  weighed  as  casein.  From 
the  first  filtrate  plus  the  wash  water  the  albumin  may  be  precipitated  by 
boiling.  The  coagulum  is  collected  on  a  Gooch  funnel,  washed  with  water 
and  alcohol  and  dried  as  before. 


MILK.  297 

It  is  also  possible  to  determine  the  total  nitrogen  by  the  Kjeldahl 
method,  and  multiply  this  by  the  factor  6.25  to  obtain  corresponding  total 
protein.     This  gives  a  fairly  good  control. 

MILK  PRESERVATIVES. 

Milk  shippers  and  dealers  often  attempt  to  keep  milk  from  spoiling— 
turning  sour  usually — by  the  addition  of  some  anti-ferment  substance. 
The  propriety  of  such  an  addition  has  been  much  discussed.  In  general 
the  use  of  food  preservatives  cannot  be  too  strongly  condemned,  as  the 
consumer  has  the  right  to  insist  on  fresh,  wholesome  food,  or  food  kept 
sterile  through  previous  heating.  Boric  acid,  salicylic  acid,  formaldehyde, 
sulphites  and  all  similar  bodies  interfere  more  or  less  strongly  with  the 
digestive  functions  when  taken  into  the  stomach.  In  the  case  of  milk  it 
is  sometimes  a  question  of  the  lesser  evil ;  the  trace  of  formaldehyde 
required  to  effectually  preserve  it  from  acid  fermentation  is  very  small. 
If  no  more  than  this  minimum  is  used  it  is  not  likely  that  the  harm  from 
using  it  would  be  very  great,  if  at  all  noticeable.  The  use  of  such  milk 
is  probably  preferable  to  that  of  the  sour,  unpreserved  milk  often  used  by 
children  in  the  poorer  quarters  of  our  cities. 

MOTHER'S  MILK. 

We  turn  now  to  a  short  discussion  of  the  chief  points  of 
difference  between  mother's  milk  and  cow's  milk,  which  is  a 
subject  of  great  practical  importance.  Success  in  substituting" 
cow's  milk  for  mother's  milk  in  the  feeding  of  small  children 
depends  very  largely  on  the  extent  and  accuracy  of  our  knowl- 
edge here.  It  is  a  singular  fact  that  we  know  much  less  about 
the  chemistry  of  human  milk  than  we  know  of  other  milks, 
and  this  is  in  part  due  to  the  difficulty  in  securing  a  perfectly 
normal  secretion  for  analysis. 

Because  of  the  presence  of  certain  salts  milk  has  a  so-called 
amphoteric  reaction,  that  is  it  shows  an  acid  behavior  with 
blue  litmus  and  an  alkaline  with  red.  In  mother's  milk  the 
alkaline  reaction  is  stronger  than  in  cow's  milk,  but  the  at- 
tempts to  determine  it  by  titration  with  the  usual  indicators 
lead  to  results  of  relatively  little  value  because  of  the  disturb- 
ing action  of  the  proteins  present.  The  salts  of  mother's  milk 
are  lower  than  in  cow's  milk. 

Analyses.     The  analysis  of  human  milk  seems  to  present 


298  PHYSIOLOGICAL    CHEMISTRY. 

several  points  of  difficulty  and  the  pul)lishetl  results  do  not 
show  very  good  agreement.  The  separation  of  the  proteins 
offers  the  greatest  difficulty,  as  the  simple  and  accurate  meth- 
ods employed  in  the  analysis  of  cow's  milk  fail  to  give  ec[ually 
satisfactory  results  when  applied  to  mother's  milk.  The  ex- 
planation of  this  will  be  given  below.  There  are  variations 
in  the  composition  of  human  milk  as  in  that  from  other  species, 
but  average  values  are  about  as  given  below  : 

Water    87.5 

Fat   3-8 

Casein 1.6 

Albumin    0.5 

Sugar    6.2 

Salts    0.4 

loo.o 

This  analysis  must  be  accepted  as  representing  the  facts 
only  in  a  general  way.  Indeed,  some  authors  go  so  far  as  to 
assert  that  no  mean  value  for  woman's  milk  is  possible,  as  the 
variations  from  individual  to  individual  are  too  great  to  per- 
mit an  average  result  to  have  any  legitimate  meaning.  This 
much,  however,  is  well  established :  the  fat  in  woman's  milk 
is  not  greatly  different  in  amount  from  that  in  cow's  milk; 
the  sugar  is  about  fifty  per  cent  higher  in  the  mean ;  the  salts 
are  lower,  sometimes  as  little  as  0.2  or  0.3  per  cent  of  ash 
being  found;  the  total  proteins  are  about  half  as  much  as  in 
cow's  milk.  But  as  to  the  relation  of  the  casein  to  the  albu- 
min and  as  to  the  nature  of  the  casein  itself,  the  greatest  diver- 
gence of  views  exists.  Some  analysts  have  actually  found 
more  albumin  than  casein  as  a  result  of  experiments.  This 
is  probably  due  to  the  employment  of  a  faulty  method  for  the 
precipitation  of  the  casein ;  it  has  been  pretty  well  established 
that  the  conditions  of  precipitation  or  coagulation  are  entirely 
different  from  those  obtaining  for  cow's  milk  It  is  indeed 
likely  that  the  protein  called  casein  in  woman's  milk  is  quite 
distinct  from  that  of  cow's  milk.  Under  the  action  of  rennet 
the  former"  coagulates  in  fine  flakes  while  the  curd  of  cow's 


MILK.  299 

milk  as  at  first  produced  is  in  very  large  flakes.  The  two 
caseins  have  different  contents  of  sulphur  and  phosphorus  and 
give  up  their  nitrogen  in  digestion  experiments  in  different 
ways.  It  has  been  recently  suggested  that  the  product  coagu- 
lated as  casein  from  human  milk  may  contain  other  proteins 
in  sufficient  amount  to  give  it  the  peculiar  properties  noticed. 
Modified  Milk.  From  all  this  it  is  evident  that  attempts 
to  modify  cow's  milk  so  as  to  make  it  resemble  mother's  milk 
must  be  more  or  less  abortive,  as  we  are  not  able  to  duplicate 
the  unknown  proteins  in  the  human  secretion.  However, 
many  suggestions  have  been  made  in  this  direction  and  the 
line  followed  is  essentially  this :  Cow's  milk  is  first  diluted 
with  an  equal  volume  of  water  or  whey  to  reduce  the  proteins 
to  the  proper  percentage  amount.  Then  a  certain  volume  of 
cream  is  added  to  restore  the  fat,  and  enough  milk  sugar  or 
cane  sugar  to  bring  that  constituent  up  to  about  6  per  cent. 
Unfortunately,  the  addition  of  fat  is  uncertain  because  of  the 
great  variations  in  market  cream.  Good  cream  should  con- 
tain at  least  20  per  cent  of  fat,  but  is  usually  much  inferior 
to  this  standard.  The  cream  sold  in  cities  often  contains  from 
5  to  10  per  cent  of  fat  only.  Assuming,  however,  a  cream 
containing  20  per  cent  of  fat,  3.5  per  cent  of  casein  and  3.5 
per  cent  of  sugar,  and  taking  the  gram  and  cubic  centimeter 
as  equivalent  for  our  purpose,  the  following  illustration  will 
serve  as  an  example  of  such  a  modification.  Starting  with 
average  market  milk  of  the  composition  given  some  pages 
back,  500  cc.  may  be  mixed  with  water,  cream  and  sugar  to 
Sfive  a  result  as  follows : 


Fat 

Sugar.  .. 
Proteins 


In  qoo  cc.  of  Market        looo  cc.  Contains,  approximately,  after  addition 
njjjjj_  of  400  cc.  of  Water,  loo  cc.  of  Cream, 

I  35  gm.  of  IMlk  Sugar. 


17-5  gm. 
22.5 


37.5  gm.  3.8  per  cent. 

61.0  I  6.1 


19.5  I  23.0  I  2.3 


Salts 1  3.5  4.0  i  0.4 

This  mixture  has  a  percentage  composition  pretty  close  to  that 
of  mother's  milk.     Sometimes  the  dilution  is  made  with  whey 


300  PHYSIOLOGICAL    CHEMISTRY. 

in  i)lace  of  water;  the  final  result  in  this  case  is  a  product  con- 
taining^ a  little  more  protein  because  of  the  content  of  albu- 
min in  the  whey. 

Another  important  distinction,  however,  must  not  be  lost 
sight  of.  While  the  milk  of  the  cow  is  sterile  when  it  leaves 
the  udder  it  takes  up  from  the  hands  of  the  milker  or  from 
the  air  a  large  number  of  bacteria  which  speedily  increase  to 
give  a  content  of  millions  to  the  cubic  centimeter.  Most  of 
these  are  doubtless  harmless  and  have  no  bad  effect  on  the 
milk ;  of  others  this  cannot  be  said,  as  their  presence  soon  leads 
to  changes  in  the  milk  which  may  render  it  absolutely  unfit 
for  use  as  an  infant  food.  ]\Iother  s  milk  is  and  remains  ster- 
ile and  is  therefore  free  from  this  danger.  It  must  be  re- 
called further  that  the  bactericidal  behavior  of  human  milk  is 
relatively  very  strong.  While  all  milks  seem  to  have  a  cer- 
tain content  of  bacteriolysins,  these  anti  bodies  in  mother's 
milk  are  most  potent  as  far  as  the  destruction  of  the  ordinary 
bacteria  is  concerned.  It  is  quite  likely  that  no  small  portion 
of  the  superiority  of  human  milk  as  infant  food  is  due  to  this 
observed  fact. 

All  kinds  of  milk  are  affected  to  some  extent  by  peculiar 
flavoring  or  other  accidental  substances  in  the  food  of  the 
parent  animal.  It  is  a  well-known  fact  that  cows  having 
access  to  certain  weeds  yield  a  milk  with  characteristic  taste 
and  odor.  In  the  same  way  many  substances  given  as  reme- 
dies pass  to  some  extent  into  the  milk  of  the  mother  and  may 
have  an  effect  on  the  nursing  child.  Bay  rum  used  for  bath- 
ing the  breasts  of  a  nursing  mother  has  been  known  to  pass, 
in  part  at  least,  into  the  milk  and  give  to  it  a  very  strong  odor 
and  taste. 

THE  MILK  OF  OTHER  ANIMALS. 

In  some  countries  the  milks  of  the  goat  and  the  ass  have 
economic  importance,  and  mare's  milk  is  used  by  certain  Asi- 
atic peoples  in  producing  a  fermented  beverage.  Analyses  of 
several  kinds  of  milk  are  on  record ;  some  of  these  are  given 


MILK. 


301 


in  the  following  table,  taken  mainly  from  the  Konig  com- 
pilation : 


Goat. 

Ass. 

Mare. 

Sow. 

Bitch. 

Cat. 

Sheep. 

Elephant. 

Water 

Fat 

Sugar 

86.9 

41 
4.4 

3-7 
0.9 

90.0 
1-3 
6.3 
2.1 

0.3 

90.0 
I.I 

6.7 
1.9 

0.3 

82.4 
6.4 
4.0 

6.1 
I.I 

75-4 
9.6 

3-1 

II. 2 

0.7 

81.6 

3-4 
4-9 
9-4 
0.7 

81.3 
6.8 

4-7 
6.4 
0.8 

67.0 
22.0 

7.4 

Proteins 

Salts 

3-0 

0.6 

Bimge  has  called  attention  to  a  relation  which  exists  be- 
tween the  composition  of  a  milk  and  the  rapidity  of  growth 
of  the  animal  feeding  on  it.  In  the  case  of,  the  young  of  the 
dog,  cat  and  sheep,  for  example,  the  rate  of  growth  immedi- 
ately after  birth  is  very  rapid  and  the  milk  of  the  mothers 
correspondingly  rich  in  proteins  and  calcium  phosphate.  The 
young  of  the  horse  and  ass  are  slow  growers,  that  is  a  rela- 
tively long  period  is  required  for  them  to  double  in  weight. 
These  mothers'  milks  are  low  in  proteins  and  salts,  but  rela- 
tively high  in  sugar.  In  the  human  species  these  relations  are 
even  more  pronounced. 


CHAPTER    XVII. 

THE   CHEMISTRY   OF   THE   LIVER.     BILE.     CELLS    IN 
GENERAL. 

From  the  earliest  clays  of  physiological  chemical  investiga- 
tion the  composition  of  the  liver  cells  and  the  nature  of  the 
processes  taking  place  there  have  heen  the  subject  of  many 
studies.  It  is  well  known  that  the  liver  has  a  certain  definite 
work  to  do  in  the  animal  organism  and  of  some  of  the  func- 
tions we  have  fairly  accurate  ideas.  Of  other  functions  there 
is  much  yet  in  dispute,  but  it  may  be  said  that  a  number  of 
synthetic  reactions  are  unquestionably  carried  out  through  the 
activity  of  cell  enzymes  there  formed.  Before  taking  up  the 
special  work  of  the  liver  cells  something  should  be  said  of 
the  composition  of  animal  cells  in  general. 

COMPOSITION  OF  CELLS. 

In  structure  all  animal  cells  agree  in  consisting  of  two  essen- 
tial parts,  a  nucleus  and  surrounding  protoplasm.  In  young 
cells  these  two  parts  are  usually  easily  recognized,  but  in  the 
old  cells  of  complex  structures  they  assume  various  forms, 
bearing  apparently  little  resemblance  to  the  original  type. 
Cells  are  in  general  the  center  of  the  various  chemical  reac- 
tions taking  place  in  the  body.  Some  of  these  reactions  take 
place  in  the  fluids  outside  the  cell,  but  by  the  aid  of  ferments 
of  cell  origin ;  most  reactions,  however,  seem  to  be  carried  on 
within  the  cell,  which  may  be  illustrated  by  the  familiar  con- 
version of  sugar  into  alcohol  and  carbon  dioxide  by  the  yeast 
cell.  The  enzyme  which  does  this  work  may,  however,  be 
extracted,  as  has  been  already  shown. 

Of  the  chemical  nature  of  nucleus  and  protoplasm  not  a 
great  deal  is  known.  It  is  extremely  difficult  to  isolate  orig- 
inal cells  from  their  modified  products  or  tissues  in  general, 

302 


CHEMISTRY    OF    THE    LIVER.  303 

and  a  sharp  chemical  differentiation  between  the  two  com- 
ponent parts  of  the  cells  is  not  yet  possible,  however  simple 
the  microscopic  differentiation  may  be.  But  some  points  have 
been  worked  out  and  these  will  be  briefly  referred  to. 

The  Nucleus.  The  most  important  chemical  constituent 
of  the  nucleus  is  the  complex  protein  substance  known  as 
miclcin,  already  referred  to  in  an  earlier  chapter.  Nucleins 
of  different  character  are  obtained  from  different  sources. 
These  nucleins  exist  in  combination  as  nucleo-proteids,  and  in 
turn  break  up  into  nucleinic  acids  and  a  protein  fraction,  which 
was  explained  in  the  chapter  referred  to.  The  cell  nucleus 
appears  to  consist  very  largely  if  not  wholly  of  the  nucleo- 
proteid.  On  digestion  with  pepsin  and  hydrochloric  acid  the 
nuclein  is  separated  and  may  be  purified  by  washing  with 
water,  dissolving  in  very  weak  alkali  and  reprecipitating  with 
acid.  The  pure  nuclein  is  a  white  amorphous  substance  which 
gives  the  Millon  test  and  the  biuret  test.  The  various  nuclein 
substances  in  cells  are  characterized  by  a  strong  affinity  for 
dye  stuffs,  especially  for  some  of  the  coal  tar  dyes ;  this  prop- 
erty is  utilized  in  the  microscopic  examination  of  tissues. 
Nuclein  fused  with  sodium  carbonate  and  nitrate  yields  phos- 
phate, but  heated  without  the  alkali  an  acid  residue  (meta- 
phosphoric  acid)  is  left. 

By  various  decompositions  nuclein  substances  yield  a  num- 
ber of  peculiar  basic  bodies  known  as  the  xanthine  or  purine 
bases,  which  will  be  considered  in  a  following  chapter.  The 
cell  nucleus  contains  in  combination  a  number  of  metallic  ele- 
ments among  which  iron  is  perhaps  the  most  important. 
Potassium  salts  are  present,  while  those  of  sodium  are  present 
only  in  traces,  if  at  all. 

The  Protoplasm.  This  soft  spongy  portion  of  the  cell 
consists  largely  of  water.  The  solid  part,  making  up  lO  to  20 
per  cent  usually,  contains  several  albumins  proper  and  proteids. 
Lecithin  is  an  important  and  relatively  abundant  constituent 
of  the  protoplasm.  Its  presence  seems  to  be  intimately  asso- 
ciated with  phenomena  of  reproduction  and  building  up  of 


304  PHYSIOLOGICAL    CHEMISTRY. 

new  tissues.  The  chemistry  of  lecithin,  as  a  complex  fat.  has 
been  explained  already.  Glycogen  and  cholesterol  are  also 
constant  constituents  of  cells,  but  of  their  uses  there  nothing 
definite  is  known. 

FUNCTIONS  OF  THE  LIVER  CELLS. 

The  anatomical  location  of  the  liver  gives  it  a  most  im- 
portant relation  to  the  other  organs  of  the  body.  With  the 
exception  of  the  fats  most  of  the  products  of  the  digestion  of 
foods  pass  through  the  portal  vein  into  the  liver  and  there 
undergo  certain  preparatory  changes.  Substances  not  true 
foods  take  the  same  course  and  many  toxic  bodies,  metallic 
and  alkaloidal,  find  a  resting  place  in  the  liver.  In  toxico- 
logical  examinations  the  liver,  after  the  stomach,  is  the  most 
important  organ  for  analysis. 

Not  only  are  the  fundamental  food  stuffs,  the  proteins  and 
the  carbohydrates,  worked  over  and  more  or  less  altered  in 
the  liver,  but  partly  metabolized  products  seem  to  be  further 
changed  in  passing  through  this  organ  and  are  there  brought 
into  a  condition  for  final  excretion.  The  evidences  that  such 
reactions  take  place  have  been  worked  out  in  a  number  of  cases 
experimentally  and  will  be  referred  to  below. 

Composition  of  the  Liver.  We  have  here  the  materials 
found  in  cells  in  general  and  also  others  having  special  func- 
tions. The  protein  substances  separated  belong  to  several 
groups;  albumin,  globulin  and  a  nucleo-proteid  have  been  rec- 
ognized. Iron  exists  in  combination  with  several  of  these 
protein  bodies.  One  of  these  is  known  as  ferratin  and  con- 
tains the  iron  in  complex  combination ;  others  appear  to  be 
albuminates  in  which  the  iron  is  more  readily  recognized. 

Next  to  the  proteins  the  fats  are  relatively  abundant  in  the 
liver  and  may  amount  to  3  or  4  per  cent  by  weight  normally. 
Pathologically,  by  fatty  degeneration,  or  by  filtration  from 
other  tissues,  the  fat  may  be  greatly  increased,  even  to  30  or 
35  per  cent  of  the  weight  of  the  whole  organ.     The  liver  fat 


CHEMISTRY    OF    THE    LIVER.  305 

is  usually  comparatively  soft,  but  that  formed  in  some  degen- 
erations is  harder. 

The  proportion  of  lecithins  in  the  liver  is  variable  and  is 
ordinarily  below  the  true  fats.  The  average  amount  is  from 
2  to  3  per  cent.  It  has  a  more  important  function  to  perform 
than  have  the  fats  proper,  since  it  is  found  by  experiment  that 
in  starvation  the  lecithin  fat  is  the  last  to  disappear.  The 
ether  extract  of  the  organ  in  this  case  is  largely  lecithin. 

The  most  important  substance  found  in  the  liver  is  probably 
glycogen,  which  is  a  transformation  product  and  variable  in 
quantity.  The  amount  present  at  any  one  moment  depends 
on  the  carbohydrate  consumption  and  the  time  which  has 
elapsed  since  a  meal.  It  may  be  as  high  as  15  per  cent  of 
the  whole  weight  of  the  organ  or  may  run  down  to  a  fraction 
of  I  per  cent,  after  fasting  or  after  the  performance  of  hard 
work.     The  formation  of  glycogen  will  be  discussed  below. 

The  liver  consisting  largely  of  cells  in  rapid  state  of  change 
furnishes  a  relatively  large  amount  of  the  so-called  nitrog- 
enous extractives.  These  include  the  xanthine  and  related 
bodies,  urea,  uric  acid,  leucine,  cystin  and  other  substances 
representing  certain  stages  in  metabolism.  The  total  amount 
of  these  compounds  present  at  any  one  time  is  very  small,  and 
probably  not  over  0.5  per  cent  of  the  dried  organ;  but  even 
this  small  amount  is  important,  as  will  appear  below. 

We  have  finally  several  mineral  substances  present.  These 
include  essentially  the  chlorides  and  phosphates  of  the  alkali. 
and  alkali  earth  metals  with  some  iron  compounds.  Of  the 
latter  those  with  proteins  have  been  mentioned  above,  but  other 
iron  salts  are  present  and  the  quantity  may  be  increased  by 
the  administration  of  inorganic  substances  as  remedies.  In 
normal  conditions  the  iron  content  is  extremely  variable. 
The  amount  may  be  accurately  determined  only  after  washing 
out  the  blood  (containing  hemoglobin)  by  aid  of  salt  solution 
of  proper  strength,  about  0.9  per  cent.  Recent  investigations 
have  shown  that  in  the  livers  of  women  the  iron  varies  from 
0.05  per  cent  to  0.09  per  cent  of  the  dry  substance,  while  in 


306  PHYSIOLOGICAL    CHEMISTRY. 

men  the  content  is  more  irregular,  running-  from  0.05  per  cent 
to  0.37  per  cent.  The  amount  seems  to  increase  ^vith  age.  but 
no  explanation  for  the  variations  can  be  given.  In  children 
and  very  young  animals  the  content  is  also  high.  It  sinks,  and 
rises  again,  later  in  life.  In  addition  to  the  iron  a  trace  of 
copper  is  said  to  be  always  present  and  may  have  some  physio- 
logical function.  Other  metals  occasionally  found  are  prob- 
ably of  accidental  occurrence,  as  the  liver  retains  such  foreign 
substances  through  a  long  period. 

CHEMICAL  CHANGES  IN  THE  LIVER. 

In  recent  years  much  has  been  written  on  this  obscure  but 
highly  important  topic.  ^lany  of  the  changes  taking  place  in 
the  liver  come  under  the  head  of  fermentations,  enzymic  reac- 
tions. Hofmeister  has  pointed  out  that  there  are  at  least 
eleven  of  these  in  play.  He  mentions  a  proteolytic  and  a 
nuclein-splitting  ferment,  one  which  splits  off  ammonia  from 
amino  acids,  a  rennet  ferment,  a  fibrin  ferment,  an  autolysing 
ferment,  a  bactericidal  ferment,  an  oxydase,  a  lipase,  a  mal- 
tase  and  a  glucase.  We  have  in  addition  to  these  reactions, 
which  result  in  general  in  the  breaking  down  of  molecules,  a 
number  of  others  which  are  synthetic  in  their  nature.  A  brief 
study  of  what  is  known  of  all  these  changes  is  sufficient  to 
indicate  the  immense  importance  of  the  liver  in  the  metabolic 
phenomena  of  the  body. 

CARBOHYDRATE  CHANGES. 

These  reactions  will  be  considered  first  because  they  have 
been  the  most  thoroughly  studied  and  also  because  of  their 
intrinsic  importance. 

Glycogen  Formation.  It  was  long  ago  established  that 
the  food  carbohydrates  after  digestion  reach  the  circulation 
almost  exclusively  by  way  of  the  portal  vein  and  the  liver. 
In  the  normal  food  of  man  and  the  herbivora  the  carbohydrate 
food  is  usually  starch  and  this  becomes  dextrin,  maltose  and 
finally  glucose  before  absorption.     As  no  marked  accumula- 


CHEMISTRY    OF    THE    LIVER.  3O7 

tion  of  the  sugar  takes  place  in  the  blood  after  a  meal  it  must 
follow  that  it  or  some  derived  reserve  product  must  be  tem- 
porarily retained  somewhere.  The  place  of  this  retention  is 
the  liver  and  the  form  in  which  the  sugar  is  held  is  glycogen. 
The  chemical  reactions  of  glycogen  have  been  discussed  in  the 
chapter  on  the  carbohydrates,  but  in  this  place  other  relations 
must  be  considered.  No  simple  answer  can  be  given  to  the 
question  as  to  the  method  of  formation  of  glycogen  from 
sugar.  Although  the  formula  is  commonly  written  CcHi^Og, 
it  is,  like  common  starch,  certainly  a  multiple  of  this.  Hence 
a  simple  equation  connecting  glucose  and  glycogen  of  the 
form 

GHi^Oe  —  H.O  1=  CeH.oOs 

is  not  strictly  correct.  Besides,  several  other  facts  appear 
which  complicate  the  problem.  While  glucose  is  ordinarily 
the  sugar  which  passes  through  the  portal  vein,  other  sugars 
are  also  consumed  and  in  the  digestive  process  do  not  become 
changed  to  glucose.  From  cane  sugar  we  have  some  fructose 
and  from  milk  sugar  some  galactose,  and  with  these  in  the 
food  it  appears  that  glycogen  is  still  formed.  Moreover  it 
has  been  shown  that  substances  not  carbohydrate  at  all  may 
give  rise  to  glycogen.  Animals  have  been  starved  until  the 
liver  was  practically  free  from  glycogen  (as  known  by  pre- 
vious trials  with  other  animals)  and  then  fed  on  fibrin  or 
washed  out  lean  meat.  On  killing  the  animals  a  short  time 
later  a  store  of  glycogen  was  found  in  the  liver,  indicating  its 
formation  from  something  in  the  protein.  With  such  facts 
in  mind  it  is  not  possible  to  form  any  simple  theory  of  the 
production  of  the  reserve  substance.  From  the  sugars  it  is 
likely  that  some  such  reaction  takes  place  as  occurs  in  the 
formation  of  starch  in  plants.  The  carbohydrate  built  up  in 
the  plant  from  water  and  carbonic  acid  is  a  sugar  and  this  is 
transformed  by  some  enzymic  reaction  into  starch  as  a  reserve 
material.  The  mechanism  of  this  change,  however,  is  quite 
obscure. 

Attempts  have  been  made  to  connect  the  formation  from 


308  PHYSIOLOGICAL    CHEMISTRY. 

proteins  with  the  sug'ar  group  of  the  gkico-proteids,  but  casein 
and  gelatin  fed  to  animals  lead  also  to  production  of  glycogen, 
and  these  bodies  in  pure  condition  do  not  furnish  a  sugar  com- 
plex by  laborator}^  treatment.  In  addition  to  this  it  is  im- 
possible that  the  sugar  group  could  be  abundant  enough  in  the 
other  common  proteins  to  account  for  the  large  amount  of 
glycogen  which  may  be  formed  by  protein  diet.  These  facts 
lead  to  the  view  that  a  synthesis  must  be  concerned  in  the 
reaction.  Such  protein  derivatives  as  leucines,  the  hexone 
bases  and  other  bodies  have  been  thought  of  as  leading  pos- 
sibly to  the  end,  but  direct  experiments  with  animals  have 
given  no  satisfactory  proof  of  such  a  hypothesis.  For  the 
present,  therefore,  the  method  of  production  from  proteins 
must  be  left  without  explanation. 

A  diet  of  fat  leads  also  to  glycogen  accumulation  or  forma- 
tion in  small  amount,  according  to  some  recent  observations. 
This  latter  reaction  requires  some  kind  of  an  oxidation  and  is 
more  difficult  of  explanation  than  the  other.  It  must  be  re- 
membered that  an  accumulation  of  glycogen  may  follow  from 
diminished  destruction  as  well  as  from  increased  production, 
and  where  the  amount  in  question  is  small,  an  apparent  in- 
crease may  be  traced  to  errors  of  observation  or  experiment. 
In  a  mixed  diet  it  is  practically  impossible  to  trace  the  effect 
of  any  one  substance.  The  behavior  of  pentoses  is  an  illus- 
tration ;  according  to  the  statements  of  some  authors  these 
carbohydrates  increase  glycogen.  It  may  be,  however,  that 
they  simply  beha^•e  as  sparers  of  glycogen  by  undergoing  oxi- 
dation, which  otherwise  the  glycogen  would  have  to  undergo. 

Not  all  the  carbohydrate  reaching  the  portal  vein  is  trans- 
formed in  the  liver;  apparently  only  a  certain  portion  is  so 
changed,  while  the  excess  is  stored  up  temporarily  in  other 
organs.  This  is  evident  from  the  fact  frequently  observed  in 
animal  experiments  that  the  amount  of  glycogen  in  the  liver 
is  below  what  should  be  expected  from  the  food  when  this  is 
excessive  in  carbohydrates.  With  ordinary  or  deficient  feed- 
ing the  liver  doubtless  is  able  to  store  as  glycogen  all  the  sugar 


CHEMISTRY    OF    THE    LIVER.  3O9 

conveyed  to  it,  but  an  excess  must  find  lodgment  elsewhere. 
The  muscles  undoubtedly  receive  the  greater  share  of  this 
excess.  In  extreme  cases  the  liver  may  hold  200  grams  of 
glycogen,  which  w-ould  correspond  to  the  same  weight  of 
starch. 

Glycogen  Destruction.  This  stored  up  glycogen  disap- 
pears in  normal  conditions  gradually  after  its  accumulation; 
the  disappearance  is  hastened  by  work  or  by  lowering  of  tem- 
perature, showing  that  it  may  be  called  upon  for  supply  of 
heat  as  well  as  for  direct  mechanical  work.  Before  being 
utilized,  however,  for  these  purposes  the  glycogen  must  be 
thrown  back  into  the  form  of  sugar;  how  this  is  done  is  still 
a  matter  of  discussion.  According  to  one  view  the  action  is 
a  "  vital  "  one,  depending  on  the  life  of  the  cells  of  the  liver 
themselves ;  by  another  view  this  conversion  is  wholly  enzy- 
mic,  a  peculiar  ferment  bringing  about  the  change  during  life 
as  well  as  post-mortem.  This  question  has  lost  much  of  its 
importance  since  the  work  of  Buchner  on  the  zymase,  or  en- 
zyme of  yeast  active  in  alcohol  formation,  as  we  now  know 
that  enzymes  are  present  where,  by  earlier  methods  of  experi- 
ment, they  were  supposed  to  be  absent. 

The  more  recent  careful  experiments  seem  to  show  beyond 
question  that  a  true  glycogen-splitting  ferment  is  present.  By 
proper  manipulation  the  cell  effect  may  be  excluded,  while 
that  of  the  ferment  is  left  intact.  This  may  be  accomplished 
in  the  following  way:  The  fresh  organ  is  washed  free  from 
blood  by  forcing  water  through  the  portal  vein  until  that 
escaping  by  the  hepatic  veins  is  clear  and  colorless.  The  liver 
is  then  chopped  fine  and  allowed  to  stand  a  day  in  a  large 
excess  of  alcohol  for  dehydration.  The  alcohol  is  poured  off, 
the  residue  pressed,  dried  at  a  low  temperature  and  ground  to 
a  powder.  In  this  form  it  is  suitable  for  extraction  with 
something  which  does  not  interfere  with  enzymic  power,  but 
which  prevents  bacterial  or  other  cell  activity.  For  this  pur- 
pose chloroform  water,  or  solutions  of  sodium  '  fluoride  have 
been  used.     A  good  extracting  mixture  may  contain  in   100 


3IO  PHYSIOLOGICAL    CHEMISTRY. 

cc.  of  water  0.2  gm.  of  sodium  fluoride  and  0.9  gm.  of  sodium 
chloride.  The  HYer  powder  is  exhausted  with  such  a  sokition 
at  a  temperature  of  38°  and  the  filtered  liquid  obtained  may 
be  used  in  two  ways.  On  standing,  the  sugar  in  the  solution 
increases  while  the  glycogen  decreases.  In  addition,  if  pure 
glycogen  be  added  to  such  an  extract  it  is  found  also  to  dimin- 
ish with  corresponding  increase  of  sugar.  It  is  further  found 
that  boiling  the  fluoride  extract  destroys  all  converting  power, 
which  fact  speaks  likew^ise  for  enzyme  action. 

The  glycogen-converting  power  of  solutions  made  as  above 
is  considerable  and  sufficient  to  fully  account  for  the  post- 
mortem increase  of  sugar  always  found  in  the  liver.  By  ex- 
tracting not  the  whole  liver  but  portions  it  is  possible  to  com- 
pare the  distribution  of  the  ferment.  Experiments  made  with 
this  end  in  view  have  shown  that  this  is  pretty  uniform.  By 
following  the  same  general  method  Pick  has  compared  the 
ferment  activity  of  the  liver  with  that  of  other  organs  where 
glycogen  may  be  stored.  In  such  experiments  the  diastatic 
action  of  the  liver  has  been  found  to  be  in  excess  as  should 
be  expected,  since  this  is  the  organ  wdiere  the  greatest  accumu- 
lation normally  takes  place.  This  normal  conversion  of  gly- 
cogen by  the  liver  ferment  is  interfered  with  by  various  sub- 
stances which  may  be  taken  as  remedies ;  quinine  salts  seem 
to  be  especially  active. 

AUTOLYTIC  FERMENTATION. 

The  liver,  or  other  organ,  removed  from  the  body  and  left 
to  itself  speedily  undergoes  a  change.  Unless  precautions  are 
taken  to  prevent  it  the  bacterial  decomposition  may  become 
pronounced  and  obscure  other  reactions.  Some  years  ago 
Salkowski  gave  the  name  auto-digestion  to  the  fermentations 
taking  place  in  the  liver,  in  which  a  change  in  the  nitrogenous 
constituents  is  mainly  involved.  Other  chemists  follow-ed  the 
subject  further,  taking  precautions  to  exclude  all  bacterial  in- 
fluences, and  have  brought  to  light  a  number  of  very  peculiar 
reactions  which  follow^  from  the  presence  of  ferments  in  the 


CHEMISTRY    OF    THE    LIVER.  3  I  I 

organs.  The  name  autolysis  has  been  given  to  these  self- 
digestion  reactions  in  general.  They  are  not  confined  to  the 
liver,  but  are  observed  in  all  organs.  An  enormous  literature 
has  accumulated  already  on  this  topic,  because  it  has  great 
practical  as  well  as  scientific  importance.  In  these  spontane- 
ous digestions  various  products  are  formed,  some  of  which 
are  volatile;  a  general  softening  of  the  tissues  concerned  may 
also  take  place  and  the  sum  of  these  changes  is  important  in 
bringing  about  the  difference  between  fresh  meat,  and  stored, 
"  ripe "  or  "  hung "  meat,  for  example.  While  bacteria 
play  an  important  part  in  curing  meat  it  is  well  known  that 
changes  go  on  within  the  tissues  which  cannot  be  due  to  bac- 
terial action.  These  are  the  autolytic  changes  which  were 
first  clearly  followed  in  the  liver,  and  which  will  be  here  briefly 
discussed. 

The  Production  of  Organic  Acids.  This  is  one  of  the 
simplest  phenomena  observed.  If  the  livers  of  dogs  or  other 
animals  are  carefully  removed  and  kept  under  chloroform  or 
toluene  a  gradual  gain  in  acidity  is  observed.  The  liver  must 
be  minced  before  being  covered  with  the  protecting  liquid. 
The  autolysis  in  this  case  is  slow,  weeks  or  months  being  re- 
quired to  show  any  large  amount  of  acid.  The  best  tempera- 
ture for  the  experiment  is  38-40°  C.  Instead  of  employing 
antiseptics  it  is  possible  with  care  to  remove  and  store  the 
liver  in  sterile  jars  aseptically.  Under  these  conditions  the 
spontaneous  change  is  very  rapid,  more  acid  being  formed  in 
one  day,  ordinarily,  than  after  a  month  of  the  antiseptic  treat- 
ment. By  making  several  pieces  of  the  liver  on  removal  from 
the  animal,  putting  each  in  a  separate  jar  and  testing  one  from 
time  to  time,  it  is  possible  to  follow  the  course  of  the  autolysis. 
Among  the  acids  produced  formic,  acetic,  fermentation  lactic 
and  paralactic,  butyric  and  succinic  have  been  recognized.  In 
experiments  described  by  Magnus-Levy  the  total  acid  formed 
in  one  day  in  100  grams  of  liver,  by  the  aseptic  treatment,  may 
correspond  to  over  20  cc.  of  normal  alkali.  If  this  were  cal- 
culated as  lactic  acid  it  would  amount  to  1.8  gm.     The  rela- 


312  PHYSIOLOGICAL    CHEMISTRY. 

tion  between  the  volatile  and  non-volatile  acids  varies  with 
the  animal,  but  not  reg-ularly. 

It  is  not  possible  to  trace  exactly  the  source  of  all  these 
acids,  but  apparently  they  come  in  part  from  a  decomposition 
of  the  sugar  of  the  liver,  since  this  is  found  to  decrease  as  the 
acid  increases.  Lactic  acid  may  be  formed  first  from  sugar 
and  butyric  acid  from  the  lactic  as  in  the  bacterial  fermenta- 
tions. The  appearance  of  hydrogen  and  carbon  dioxide  at 
the  same  time  favors  this  view. 

The  Alteration  in  the  Proteins.  When  subjected  to  asep- 
tic auto-digestion,  or  to  the  same  digestion  with  chloroform 
or  toluene,  the  protein  substances  gradually  break  down  into 
simpler  products.  Among  these  the  amino  acids  may  be  most 
readily  recognized;  there  is  also  an  increase  in  the  nitrogen 
which  may  be  distilled  off  with  magnesia.  The  behavior  here 
is  somewhat  similar  to  that  which  follows  in  acid  hydrolysis 
of  the  proteins,  or  which  occurs  in  prolonged  boiling  with 
water  under  pressure;  in  both  cases  a  kind  of  hydrolysis  re- 
sults and  this  may  be  what  takes  place  in  auto-digestion. 

In  prolonged  aseptic  auto-digestion  of  the  liver  very  con- 
siderable quantities  of  leucine  and  tyrosine  are  formed ;  on  the 
outer  surfaces,  where  evaporation  can  take  place,  the  latter 
may  even  separate  in  crystalline  bunches  easily  recognized. 
The  hexone  bases  and  bodies  of  the  xanthine  group  also  result 
but  not  always  in  ver}-  great  quantities. 

Pathological  Importance.  This  possibility  of  self-diges- 
tion in  the  liver  and  other  organs  may  help  explain  some  of 
the  phenomena  observed  in  pathological  conditions.  The 
acids  found  sometimes  in  the  urine  as  well  as  the  leucine  and 
tyrosine  have  usually  been  traced  to  the  liver.  These  experi- 
ments show  the  rapidity  with  which  such  products  may  be 
formed  by  a  degenerative  process.  Pathologically  the  urine 
sometimes  shows  a  very  high  reducing  power  which  cannot 
be  associated  wnth  sugar  or  uric  acid  or  creatinine.  The 
liquid  formed  in  the  liver  autolysis  is  always  strongly  reducing 
in  action  and  this  may  suggest  an  explanation  for  the  obser- 
vation on  the  urine. 


CHEMISTRY    OF    THE    LIVER.  3I5 

Bactericidal  Products.  It  is  worthy  of  note  that  in  these 
autolytic  decompositions  substances  are  formed  which  have  a 
marked  bactericidal  action.  This  has  been  shown  in  many 
ways  and  the  suggestion  appears  reasonable  that  in  the  con- 
tinuous breaking  down  processes  going  on  in  various  organs 
we  have  some  of  the  factors  of  natural  immunity.  These 
autolytic  products  must  not  be  confounded  with  the  alexins 
already  referred  to.  In  a  few  experiments  on  record  injec- 
tions with  pressed  out  juice  from  autolyzed  organs  have  been 
sufficient  to  prevent  death  in  small  animals  infected  with  viru- 
lent cultures.  The  bactericidal  action  of  the  fresh  liver  or 
other  organ  is  comparatively  slight. 

OTHER  FERMENT  ACTIONS. 

Other  ferments  present  in  the  liver  have  not  been  very 
thoroughly  studied.  The  presence  of  a  fat-splitting  ferment 
or  lipase  has  been  shown,  but  little  is  known  of  the  extent  of 
its  action  in  the  body.  The  oxidase  ferments  are  better 
known  and  the  action  of  liver  extracts  in  bringing  about  oxi- 
dations of  various  organic  substances  has  been  studied  with 
the  object  of  throwing  some  light  on  normal  oxidations  in  the 
body.  How  many  of  these  oxidizing  ferments  the  liver  may 
contain  is  of  course  not  known.  The  reaction  thus  far  the 
most  carefully  studied  is  that  between  water  extracts  of  the 
liver  and  salicylic  aldehyde.  In  the  process  this  becomes  sali- 
cylic acid. 

The  action  of  a  liquid  obtained  by  pressing  the  minced  liver 
ground  up  with  sand  has  been  studied  with  reference  to  its 
power  of  hydrolyzing  certain  esters.  Amyl  salicylate  seems 
to  be  readily  split  by  this  liver  juice.  The  reaction  points  to 
the  presence  of  a  lipase-like  ferment  which  doubtless  has  the 
power  of  splitting  other  bodies  of  this  type.  The  boiled  liquid 
is  without  the  ester-splitting  power.  It  has  been  found  fur- 
ther that  the  active  element  can  be  completely  salted  out  by 
addition  of  ammonium  sulphate  to  saturation,  and  it  may  be 
precipitated  by  addition  of  a  strong  solution  of  uranium 
acetate. 


314  PHYSIOLOGICAL    CHEMISTRY. 

THE   BEHAVIOR   OF    THE    LIVER    WITH    POISONS. 

The  fact  has  been  referred  to  already  that  many  nietalhc 
and  some  organic  substances  combine  with  the  Hver  cells.  All 
this  has  a  practical  bearing  on  toxicological  investigations,  in 
which  experience  has  shown  the  importance  of  including  the 
liver  in  the  analytical  tests.  Recent  experiments  have  thrown 
some  light  on  the  question  of  the  manner  of  combination  of 
poisons.  Corrosive  sublimate,  for  example,  fed  in  very  small 
portions  to  dogs  was  found  later  by  post-mortem  examina- 
tions in  the  globulin  fraction  of  the  liver  extract.  The  fixa- 
tion of  arsenic  is  different;  it  combines  with  a  nuclein  substance 
and  in  very  stable  form,  which  explains  the  practical  difficulty 
of  separating  this  substance  in  forensic  investigations. 

Experiments  have  also  been  published  showing  the  behavior 
of  small  doses  of  morphine  sulphate  and  strychnine  sulphate 
in  the  liver.  It  appears  that  the  retaining  power  of  the  liver 
for  these  poisons  is  relatively  large  when  they  are  administered 
by  the  mouth  or  injected  into  the  portal  vein.  The  retention 
of  the  alkaloids  by  the  organ  has  been  experimentally  shown. 
Such  observations  have  an  important  bearing  in  explaining  the 
fact  that  many  poisons  are  far  more  active  when  injected  hy- 
podermically  than  when  given  through  the  stomach.  This 
seems  to  be  true  of  many  substances  besides  the  metallic  poi- 
sons and  the  alkaloids.  The  phenols,  for  example,  are  like- 
wise retained  to  a  marked  extent  by  the  liver. 

SYNTHETIC   PROCESSES   IN    THE   LIVER. 

It  has  long  been  known  that  the  liver  is  the  seat  of  the  for- 
mation of  a  large  number  of  metabolic  products  some  of 
which  involve  syntheses.  Several  of  these  reactions  may  be 
briefly  explained  in  this  place,  but  nothing  like  a  full  discus- 
sion will  be  attempted.  A  few  illustrative  cases  only  will  be 
taken  to  show  in  a  general  way  what  is  best  known  in  this 
field. 

The  Formation  of  Urea.  Of  all  the  synthetic  reactions 
known  to  occur  wholly  or  in  part  in  the  liver  this  one  has  been 


CHEMISTRY    OF    THE    LIVER.  315 

the  most  thoroughly  studied.  The  older  notion  of  the  forma- 
tion of  urea  from  the  more  complex  uric  acid  is  no  longer 
held ;  indeed  no  simple  relation  exists  between  the  two  sub- 
stances, as  it  is  pretty  well  established  that  the  destruction  of 
the  nuclein  bodies  is  mainly  responsible  for  the  uric  acid  of 
the  urine.  A  great  many  observations  unite  in  suggesting  the 
liver  as  the  organ  in  which  urea  is  most  abundantly  produced, 
and  certain  ammonium  salts  as  being  largely  or  mainly  con- 
cerned in  this  production. 

It  has  been  noted  clinically  that  in  diseases  of  the  liver  there 
may  be  at  the  same  time  a  marked  diminution  in  the  excretion 
of  urea  with  a  corresponding  increase  in  the  ammonia  excre- 
tion. It  is  also  known  that  the  administration  of  ammonium 
salts  is  not  followed  by  an  increase  of  ammonia  in  the  urine. 
Parallel  with  this  observation  we  have  the  further  one  made 
on  the  blood,  which  has  shown  that  the  fluid  of  the  portal  vein 
is  far  richer  in  ammonia  than  is  that  from  the  hepatic  vein. 
Such  observations  have  been  followed  up  by  experiments  in 
which  fresh  blood  is  forced  through  a  living  or  a  recently 
removed  liver  by  means  of  specially  constructed  apparatus. 
The  same  blood  may  be  caused  to  pass  the  liver  many  times. 
After  passing  a  few  times  and  reaching  uniformity  in  compo- 
sition various  ammonium  and  related  compounds  are  added 
to  the  blood  and  the  circulation  then  continued.  In  this  way 
the  abundant  transformation  of  ammonium  carbonate  into 
urea  is  readily  shown.  It  has  also  been  found  that  certain 
amino  acids  are  converted  rather  readily  in  going  through  the 
liver.  Experiments  have  shown  that  in  the  course  of  a  few 
hours  several  grams  of  leucine,  glycocoll  or  aspartic  acid  may 
be  transformed  into  urea  under  these  unfavorable  conditions. 

The  importance  of  this  observation  will  be  recognized.  It 
is  well  known  that  the  amino  acids  are  among  the  most  im- 
portant of  the  disintegration  products  of  the  proteins;  by 
hydrolytic  and  other  cleavage  reactions  these  amino  complexes 
result,  and  we  see  here  the  possibility  of  further  destruction 
M^ith  ultimate  formation  of  urea.     It  is  possible  that  in  this 


3l6  niVSIOLOGICAL    CHEMISTRY. 

reaction  carbamic  acid  is  the  immediate  forerunner  of  tlie  urea. 
It  may  be  formed  from  some  amino  acids  by  oxidation  proc- 
esses and  in  turn  passes  to  urea  by  this  reaction, 

NH.O  •  CO  •  NH.  +  H=  =  NH.CONIL  +  H=0, 

in  which  a  re(hicti()n  process  is  finally  in  play.     The  carbamic 

acid  may  be  produced  by  oxidation  directly  of  the  ammonium 

salt, 

NH^O  ■  CO  •  NH=  +  O  =  NH.O  ■  CONH^  +  H.O, 

SO  that  both  oxidations  and  reductions  may  be  concerned. 
However,  we  are  certain  of  the  final  result  only,  and  are  not 
able  to  o^ive  the  steps  definitely. 

The  Formation  of  Ethereal  Sulphates.  Another  reac- 
tion of  far-reaching-  importance  in  the  body  is  the  production 
of  organic  sulphates.  The  oxidation  of  the  sulphur  of  pro- 
teins leads  finally,  mainly,  to  the  formation  of  sulphuric  acid 
which  is  eliminated  in  the  urine  in  the  form  of  the  ordinary 
mineral  sulphates  and  the  ethereal  sulphates.  The  mineral 
sulphates  are  readily  formed  directly  by  combinations  in  the 
blood,  but  for  the  union  of  the  organic  groups  with  sulphuric 
acid  some  active  agent  is  required.  The  addition  seems  to 
take  place  in  the  liver  where  it  is  probable  that  the  oxidation 
of  the  sulphur-containing  complex,  furnished  by  protein  dis- 
integration, also  occurs.  This  complex  seems  to  be  cystein, 
which  is  easily  oxidized  to  cystin,  and  experiments  with  ani- 
mals have  shown  that  cystein  administered  increases  largely 
the  sulphates  of  the  urine.  Several  attempts  have  been  made 
to  determine  the  seat  of  the  reaction  by  irrigation  tests,  and 
comparatively  recently  it  has  been  shown  that  blood  containing 
phenol  and  cystin  and  led  through  the  liver,  freshly  dissected, 
discloses  a  ver}^  considerable  oxidation  of  the  sulphur  com- 
pound \\ith  production  of  aromatic  sulphate.  It  appears  that 
other  organs  are  not  much  concerned,  if  at  all,  in  the  reaction. 

The  aromatic  radicles  which  join  with  sulphuric  acid  in  this 
way  are  mainly  products  of  intestinal  putrefactive  changes, 
and  by  absorption  finally  reach  the  liver.     In  addition  to  sul- 


CHEMISTRY    OF    THE    LIVER.  31/ 

phuric  acid  glucoronic  acid  acts  to  hold  the  phenol  bodies ;  it 
is  usually  present  in  traces  in  normal  urine  and  is  often  greatly 
increased  pathologically.  The  glucoronates  may  be  formed  in 
the  liver  along  with  the  aromatic  sulphates.  In  experiments 
which  have  been  carried  out  on  the  passage  of  the  blood 
through  a  liver  the  conjugate  phenol  bodies  produced  have 
frequently  been  in  excess  of  the  amount  called  for  by  the  sul- 
phuric acid  found;  this  excess  may  correspond  in  the  main 
with  the  glucoronic  acid. 

The  Synthesis  of  Uric  Acid.  The  mode  and  place  of  the 
formation  of  uric  acid  in  the  animal  organism  have  been  the 
subjects  of  numerous  investigations.  In  birds,  serpents  and 
some  of  the  mammals  the  excretion  of  nitrogen  is  largely  in 
the  form  of  uric  acid,  and  experiments  have  shown  that  it  is, 
in  part  at  least,  of  synthetic  origin.  The  excretion  of  uric 
acid  in  birds  is  increased  by  doses  of  ammonium  salts ;  with 
the  livers  extirpated  there  is  a  decrease  in  the  elimination  of 
uric  acid  and  increase  in  excretion  of  ammonium  compounds. 
In  a  number  of  such  observations  the  liver  has  been  connected 
with  uric  acid  formation,  and  transfusion  experiments,  in 
which  blood  containing  ammonium  lactate  and  certain  other 
compounds  has  been  forced  through  the  livers  of  geese, 
pointed  to  the  same  kind  of  a  synthetic  conversion.  For  the 
higher  animals,  however,  a  different  formation  has  usually 
been  assumed,  the  oxidation  of  the  xanthine  bodies  coming 
from  the  breaking  down  of  nucleins  being  looked  upon  as  the 
principal  formative  reaction. 

Comparatively  recent  experiments  by  several  authors  sug- 
gest synthetic  reactions  as  likewise  possible.  Wiener,  for  ex- 
ample, mixed  chopped  beef  liver  with  physiologic  salt  solution 
and  allowed  the  mixture  to  stand  at  the  body  temperature  an 
hour.  The  liquid  was  then  pressed  out  and  the  uric  acid  in 
it  determined  after  some  time  in  a  given  volume.  To  the 
same  volume  of  liver  extract  definite  weights  of  urea  and  vari- 
ous ammonium  and  sodium  salts  were  added  and  the  mixture 
allowed  to  stand  as  before.     In  certain  cases  a  very  marked 


3l8  PHYSIOLOGICAL    CHEMISTRY. 

increase  in  the  uric  acid  resulted,  pointing  to  the  presence  in 
the  Hver  extract  of  some  agent  capable  of  effecting  the  com- 
bination. The  best  results  were  obtained  with  dialuric  acid 
salts,  and  tartronic  acid  and  urea.  The  significance  of  these 
special  reactions  will  be  pointed  out  later,  the  fact  of  impor- 
tance here  being  the  behavior  of  the  liver  extract  in  the  com- 
bination. It  has  been  found  that  liver  extracts  have  also  the 
power  of  converting  purine  bodies  into  uric  acid.  It  is  likely 
that  the  other  organs  furnish  active  agents  capable  of  perform- 
ing the  same  work,  but  probably  in  lower  degree. 

THE  BILE. 

The  formation  of  bile  is  one  of  the  important  functions  of 
the  liver  and  the  amount  secreted  in  man  is  several  hundred 
grams  daily.  Some  of  the  uses  of  the  bile  have  been  referred 
to  in  earlier  chapters  under  the  head  of  digestion  phenomena. 
Other  functions  will  be  discussed  presently. 

AMOUNT  AND  COMPOSITION. 

The  volume  of  the  bile  secreted  seems  to  be  subject  to  varia- 
tions which  are  not  well  understood.  Through  the  aid  of  a 
biliary  fistula  it  is  possible  to  collect  the  total  excretion  in  dogs 
and  other  animals  which  are  easily  experimented  upon  and 
determine  the  rate  of  flow  and  the  whole  amount.  The  vol- 
umes reported  by  different  observers  are  not  in  good  agree- 
ment. The  amounts  secreted  by  different  animals  in  24  hours 
for  each  kilogram  of  body  weight  vary  between  12  grams  for 
the  goose  and  137  grams  for  the  rabbit.  For  man  the 
amounts  observed  have  varied  between  about  150  and  i.ooo 
grams  daily. 

The  flow  of  the  bile  is  increased,  as  far  as  volume  is  con- 
cerned at  any  rate,  by  the  administration  of  certain  remedies. 
These  are  known  as  cholagogues  and  among  them  calomel, 
certain  resins,  rhubarb  and  oil  of  turpentine  are  perhaps  best 
known.  That  the  solids  of  the  secretion  are  increased  is  a 
disputed  question.     It  is  proper  to  state  here  that  many  of  the 


CHEMISTRY    OF    THE    LIVER. 


319 


older  data  on  this  subject  were  obtained  by  methods  which  are 
open  to  serious  objection. 

Composition  of  Bile,  Ouahtatively  bile  is  characterized 
by  the  presence  of  certain  acids  and  coloring  matters  which 
are  not  found  elsewhere  in  the  body.  The  acids  are  taitro- 
cholic  and  glycocholic,  and  the  coloring  matters  are  bilirubin 
and  biliverdin,  which  have  been  referred  to  already  in  their 
relation  to  the  coloring  matter  of  blood  from  which  they  are 
derived.  In  addition  to  these  substances  several  others  are 
present  which,  while  important,  are  not  characteristic.  These 
include  cholesterol,  fats,  soaps,  inorganic  salts  and  mucin. 
The  quantitative  composition  is  extremely  variable  as  shown 
by  the  analyses  below  which  are  frequently  quoted.  The 
results  are  in  parts  per  i  ,000 : 


Water  

Solids 

Biliary  salts 

Mucine  and  pigments. 

Cholesterol 

Lecithin  

Fat 

Soaps 

Inorganic  salts 


I 

2 

860.0 

I40.C 
72.2 

26.6 

1.6 

859.2 

140.8 

91.4 

29.8 

2.6 

3-2 

9.2 

6.5 

7-7 

822.7 

177-3 

107.9 

22.1 

47-3 


10.8 


898.1 

101.9 

56.5 

14-5 

30.9 


6.2 


977-4 
22.6 
10. 1 

2-3 
0.6 
0.1 
0.1 
1-4 
8-5 


The  figures  under  i,  2,  3  and  4  were  obtained  by  analyses  of 
secretions  from  the  gall  bladders  of  persons  executed  or  acci- 
dentally killed.  They  therefore  represent  normal  products. 
No.  5  is  bile  from  a  fistula;  it  is  seen  to  be  much  poorer  in 
solids  than  the  other  biles,  which  result  is  confirmed  by  many 
other  analyses.  It  follows  from  this  that  the  secretion  in  the 
bladder  undergoes  a  concentration  by  absorption  of  water. 

Analyses  have  been  made  of  bile  from  different  animals 
with  the  object  of  connecting  composition  with  the  food  of 
the  animal  or  its  habits.  The  results  are  not  very  definite. 
Human  bile  contains  more  glycocholic  than  taurocholic  acid, 
while  in  carnivorous  mammals,  birds  and  fishes  taurocholic 
acid  is  the  more  abundant.     Hog  bile  contains  largely  glyco- 


320  PHYSIOLOGICAL    CHEMISTRY. 

cholic  acid,  but  in  ox  bile  the  relation  is  variable.  The 
amounts  of  the  pis^ments  are  small  and  not  accurately  known. 
Glycocholic  Acid.  This  is  a  complex  substance  made  up 
of  a  combination  of  glycocoll  or  glycine  with  cholalic  acid. 
The  constitution  of  the  acid  is  not  known,  but  the  empirical 
formula  C2f;H43NOo  has  been  given  to  it.  In  the  bile  it  exists 
in  the  form  of  a  sodium  or  potassium  salt,  which  is  readily 
soluble  in  water  or  alcohol.  The  free  acid  is  but  slightly  solu- 
ble; hence  the  addition  of  mineral  acids  to  bile  produces  a 
precipitate.  On  boiling  a  solution  of  glycocholic  acid  with 
weak  acids  or  alkalies  a  cleavage  follows,  and  glycocoll  and 
the  nitrogen-free  cholalic  acid  separate.  Water  is  taken  up 
at  the  same  time.  This  is  a  reaction  analogous  to  the  separa- 
tion of  glycocoll  and  benzoic  acid  from  hippuric  acid  by  the 
same  manner  of  treatment.  There  appear  to  be  several 
cholalic  acids,  but  with  the  common  one  the  reaction  would 
be  represented,  probably,  in  this  way : 

C«,H43NOo  +  H.O  =  C^HaO.NH.  +  O.MM,. 

Cholalic  Acid.  Although  many  investigations  have  been 
carried  out  with  this  substance  its  constitution  is  not  clear. 
The  above  empirical  formula,  C24H40O5,  is  that  of  a  mono- 
basic acid  to  which  ]\Iylius  has  given  this  possible  structure, 


CMz 


CHOH 
CH2OH 
CH=OH 
COOH 


The  free  acid  is  very  slightly  soluble  in  water,  but  the  alkali 
salts  are  readily  soluble.  The  free  acid  is  somewhat  soluble 
in  ether;  hence  it  is  found  as  a  decomposition  product  of  the 
bile  acids  in  the  crude  fat  extracted  from  feces.  By  oxidation 
cholalic  acid  yields  several  new  acids  which  have  been  much 
studied  with  the  hope  of  gaining  an  insight  into  the  struc- 
ture of  the  original  acid.  Among  the  various  derived  acids 
these  may  be  mentioned :  Cholcic  acid,  C23H42O4.  dchydro- 
cholcic  acid,  C.2ill-^_iO^,  cholanic  acid,  C04H34OS.     Fcllic  acid. 


CHEMISTRY    OF    THE    LIVER.  321 

C23H40O4,,  and  lithofcllic  acid,  C^oHogOj,  are  found  in  some 
kinds  of  bile. 

Taurocholic  Acid.  To  this  substance  the  empirical  form- 
ula C26H45NSOY  is  given.  With  weak  acids  it  undergoes 
likewise  a  hydrolytic  cleavage  from  which  taurin  and  cholalic 
acid  result.  Taurin  appears  to  be  aminoethylsulphonic  acid, 
C2H4NH2  "HSOg,  and  the  cleavage  would  be  represented  in 

this  way : 

GoH«NSOt  +  H.O  =  Q4H40O5  +  GH.NH.HSOa. 

The  free  acid  has  a  bitter-sweet  taste ;  it  is  much  more  soluble 
in  water  than  the  glycocholic  acid  and  somewhat  soluble  in 
alcohol.  The  free  acid  has  the  property  of  holding  glyco- 
cholic acid  in  aqueous  solution,  which  is  shown  by  the  diffi- 
culty in  precipitating  the  mixed  acids  from  ox  bile.  The  free 
acid  is  but  slightly  soluble  in  ether.  The  alkali  salts  are  solu- 
ble in  water  and  alcohol. 

Preparation  of  Acids  from  Ox  Bile.  This  may  be  illustrated  by  the  fol- 
lowing. Evaporate  200  to  300  cc.  of  the  bile  to  dryness,  or  as  near  to 
dryness  as  possible,  on  the  water-bath  with  the  addition  of  about  60  grams 
of  bone-black.  After  cooling  the  mass  rub  it  up  thoroughly,  transfer  to 
a  flask  and  extract  with  alcohol  by  heating  over  a  water-bath.  The  two 
bile  salts  are  soluble  in  the  alcohol,  while  the  mucin  and  inorganic  salts 
present  are  not.  Therefore  cool  the  extracted  mixture  and  filter.  The 
filtrate  contains  the  bile  salts  along  with  cholesterol,  some  fat  and  traces 
of  other  substances.  There  is  also  some  water  present.  Evaporate  the 
filtrate  to  dryness,  take  up  with  absolute  alcohol  and  filter  again.  Taking 
advantage  of  the  practical  insolubility  of  the  bile  salts  in  ether  they  may 
be  precipitated  in  this  way :  Add  to  the  strong  alcoholic  solution  an  excess 
of  dry  ether,  or  enough  to  cloud  the  mixture,  and  allow  to  stand.  After 
some  hours  or  days  a  crystalline  precipitate  of  the  bile  salts  separates. 
The  crystals  may  be  used  for  preparation  of  other  substances,  or  for  tests. 
In  the  mother  liquor  cholesterol  may  be  detected  by  the  tests  given  in  an 
earlier  part  of  this  work. 

Preparation  of  Glycocholic  Acid.  Use  the  larger  part  of  the  above 
described  crystalline  precipitate  for  this  purpose.  Dissolve  in  water  and 
add  enough  dilute  sulphuric  acid  to  produce  a  marked  turbidity.  Add  a 
little  ether,  shake  the  mixture  well  and  allow  to  stand  in  a  cold  place. 
The  glycocholic  acid  separates  in  the  form  of  fine  silky  needles.  Press 
out  the  mother  liquor,  redissolve  in  hot  water  and  allow  to  crystallize  a 
second  time.     A  nearly  pure  product  may  be  so  obtained. 

Preparation  of  Taurocholic  Acid.     The  separation  of  this  acid  from  the 


322 


PHYSIOLOGICAL    CHEMISTRY 


glycocholic  acid  is  extremely  difficult,  hence  in  preparing  it,  it  is  best  to 
start  with  a  bile  which  contains  essentially  only  the  one  salt.  Dog's  bile 
should  therefore  be  employed.  Treat  it  as  described  for  the  mixed  salts, 
and  decompose  finally  with  dilute  sulphuric  acid  in  presence  of  ether. 

Preparation  of  Cholalic  Acid.  Dissolve  200  grams  of  barium  hydroxide 
in  6  liters  of  water.  In  this  solution  saponify  50  gm.  of  glycocholic  acid, 
by  boiling  ten  to  twenty  hours,  replacing  the  water  lost  by  evaporation. 
Filter  hot  and  to  the  cooled  liquid  add  enough  hydrochloric  acid  to  decom- 
pose the  barium  salt.  The  cholalic  acid  separates  in  the  form  of  a  granu- 
lar precipitate.     Wash  with  water  and  crystallize  from  hot,  strong  alcohol. 

Optical  Properties  of  These  Acids.  The  three  acids  and  their  sodium 
salts  are  characterized  by  rather  strong  rotating  power,  which  under  some 
circumstances  may  be  used  for  measurement  or  identification.  The  fol- 
lowing specific  rotations  have  been  found : 


For  Aqueous  Solution. 


Wi, 


Glycocholic  acid 

sodium  salt '    24.928    [    +20.8° 

Taurocholic  acid,  sodium  salt ;       8.856        -["21.5 

Cholalic  acid,  anhydrous 

sodium  salt 19.049        -f-26.0 


For  Alcohol  Solution. 


9-504 
20. 143 
9.898 
2.942 
2.230 


W/) 


+29.0° 
+  25-7 

+  24-5 

4-47-6 

+31-4 


Chemical  Test  for  the  Bile  Salts.  The  three  acids  are  characterized 
by  giving  a  certain  reacfion  with  furfuraldehyde,  or  sugar  yielding  fur- 
furaldehyde,  in  presence  of  acid.  The  test  is  commonly  made  by  adding 
to  a  dilute  solution  of  the  salts,  say  5  cubic  centimeters,  a  few  drops  of 
a  dilute  cane  sugar  solution  and  strong  sulphuric  acid  in  volume  about 
half  that  of  the  mixture.  Let  the  acid  flow  down  the  side  of  the  test- 
tube  so  as  to  form  a  layer  below  the  lighter  liquid.  A  deep  purple  band 
appears  at  the  line  of  contact.  On  slowly  mixing  the  liquids  in  the  test- 
tube  the  color  becomes  purple  throughout.  In  this  test  any  excess  of 
sugar  must  be  avoided. 

With  a  trace  of  pure  furfuraldehyde  in  place  of  sugar  the  reaction  is 
sharper,  but  certain  proportions  must  be  observed.  A  good  mixture  is 
I  cubic  centimeter  of  weak  alcoholic  solution  of  the  bile  acid,  i  drop  of 
0.1  per  cent  furfuraldehyde  solution  and  i  cubic  centimeter  of  strong 
sulphuric  acid.  The  original  test  was  devised  by  Pettenkofer;  later  it 
was  recognized  that  the  reaction  belongs  to  the  group  of  "  furfurol "  reac- 
tions, and  the  aldehyde  was  recommended  in  place  of  the  sugar.  The 
test  cannot  be  used  with  bile  directly  because  of  the  presence  of  other 
substances,  which  would  give  a  strong  color  with  the  sulphuric  acid. 

Preparation  of  Taurin.  Use  several  hundred  cubic  centimeters  of  ox 
bile.  Add  to  it  an  excess  of  strong  hydrochloric  acid,  about  one-third 
of  the  volume  of  the  bile,  and  boil  on  the  water-bath.  A  resinous  mass 
separates  and  when  this  becomes  stringy  enough  to  solidify,  when  a  little 


CHEMISTRY    OF    THE    LIVER.  323 

is  taken  up  on  a  rod  and  allowed  to  cool,  the  reaction  has  gone  far 
enough.  Decant  from  this  mass  and  evaporate  the  liquid  resulting  until 
a  crystallization  of  salt  forms.  Filter  and  evaporate  to  a  small  volume. 
If  salt  separates  filter  again  and  pour  the  liquid  finally  into  a  large  excess 
of  alcohol.  This  causes  the  taurin  to  separate;  wash  the  crude  substance 
with  strong  alcohol,  and  recrystallize  from  hot  water.  In  a  successful 
separation  large  plates  or  prisms  of  taurin  are  obtained.  The  substance 
may  be  recognized  by  several  tests.  On  heating  it  chars  and  gives  off 
an  odor  of  sulphurous  acid.  When  fused  with  sodium  carbonate  the  sul- 
phur is  converted  into  sulphide,  from  which  hydrogen  sulphide  maj'  be 
separated  and  identified  by  the  usual  tests. 

THE  BILE  PIGMENTS. 

The  two  substances  biliverdin  and  bilirubin  are  related  to 
hematin  from  hemoglobin,  as  pointed  out  above,  and  as  may 
be  illustrated  by  these  formulas : 

Hematin   C32H32N404Fe 

Hematoporphyrin    C32H36N40g 

Bilirubin  C32H36N4O6 

Biliverdin  C32H36N4O8 

The  two  bile  pigments  are  formed  in  the  liver  and  normally, 
apparently,  only  in  the  liver,  but  by  what  kind  of  reaction  is 
not  clearly  known.  Hematoporphyrin  may  be  produced  from 
hematin  and  it  is  isomeric  with  bilirubin,  though  not  identical. 
The  relation  of  bilirubin  to  blood  is  perhaps  best  shown  by 
this  observation :  in  old  blood  extravasations  the  blood  color 
appears  to  be  gradually  decomposed  and  in  its  place  the  new 
coloring  matter  is  found,  which  was  called  hematoidin  by  its 
discoverer.     Later  studies  showed  the  identity  with  bilirubin. 

Bilirubin  is  practically  insoluble  in  water,  but  it  seems  to 
act  as  an  acid,  the  alkali  salts  of  which  are  soluble.  In  this 
form  it  exists  in  bile.  The  solution  is  reddish  yellow  and  in 
the  air  or  by  treatment  with  oxidizing  agents  it  takes  up  oxy- 
gen and  becomes  biliverdin,  which  gives  a  green  solution. 
The  bile  always  contains  the  two  pigments,  from  which  the 
greenish  yellow  color  follows.  The  amount  of  the  two  sub- 
stances in  the  bile  is  normally  very  small,  but  as  the  reactions 
are  sharp  recognition  is  easy.     The  total  weight  of  the  two 


324  PHYSIOLOGICAL    CHEMISTRY. 

pigments  produced  in  one  day  is  not  over  200  milligrams  prob- 
ably; the  physiological  meaning  of  the  formation  is  not  known. 
The  iron  of  the  original  hematin  is  largely  retained  by  the 
substance  of  the  liver  cells. 

Preparation  of  Bilirubin.  The  pigment  cannot  be  easily  obtained  from 
bile  i)ecause  of  tlie  small  amount  present,  but  may  be  obtained  from  the 
pathological  concretions  known  as  gall-stones,  which  will  be  described 
later.  Powder  several  grams  of  these  stones  from  cattle  very  fine  and 
exhaust  thoroughly  with  ether,  then  repeatedly  with  boiling  water  to  take 
out  cholesterol  and  bile  acids.  In  the  residue  the  bilirubin  exists  as  an 
insoluble  calcium  compound ;  this  is  decomposed  by  the  addition  of  a  little 
dilute  hydrochloric  acid,  after  which  what  is  left  is  washed  thoroughly 
with  hot  water,  and  then  with  alcohol  to  leave  the  pigment  in  a  still  better 
condition  for  extraction.  Finally  extract  with  chloroform  in  which  the 
substance  is  relatively  soluble.  On  evaporating  the  chloroform  crude 
bilirubin  is  secured,  which  after  washing  with  alcohol  may  be  recrystallized 
from  hot  chloroform  or  from  dimethylaniline,  in  which  it  dissolves  in 
the  proportion  of  about  i  to  100  cold  or  i  to  30  hot.  By  several  crystal- 
lizations it  is  possible  to  obtain  a  product  pure  enough  to  employ  as  a 
standard  for  spectroscopic  measurements. 

By  exposing  an  alkaline  solution  to  the  air  or  by  treating  with  a  little 
acid  and  sodium  peroxide,  bilirubin  is  converted  into  biliverdin.  The  lat- 
ter free  substance  is  not  soluble  in  water,  chloroform  or  ether. 

The  Bile  Pigment  Tests.  Some  of  these  are  extremely  delicate  and 
have  long  been  used  for  the  recognition  of  bile,  especially  in  urine.  For 
the  tests  to  be  given  the  bilirubin  alkali  in  very  dilute  solution  may  be 
used,  or  a  diluted  bile. 

Gmeun's  Test.  In  a  test-tube  take  a  few  cubic  centimeters  of  nitric 
acid  containing  some  nitrous  acid.  Over  this  pour  carefully  the  weak  bile 
solution  to  be  tested.  At  the  junction  point  colored  rings  appear  which 
result  from  the  formation  of  oxidation  products  of  the  bilirubin.  The 
colors  appear  in  this  order  from  above  down :  green,  blue,  violet,  red  and 
yellowish.  Of  these  the  green  is  the  most  characteristic ;  the  other  shades 
represent  more  advanced  stages  in  the  oxidation.  For  success  in  the  test 
the  bile  solution  must  not  be  too  strong,  and  the  amount  of  nitrous  acid 
in  the  nitric  acid  must  be  small. 

H.\mm.\rsten's  Test.  Use  as  reagent  a  mixture  of  strong  nitric  acid 
and  strong  hydrochloric  acid  in  the  proportion  of  about  I  to  50  by  vol- 
ume. This  mi.xture  must  stand  some  time  before  use,  or  until  it  becomes 
yellow.  It  keeps  a  long  time.  For  the  practical  test  mix  i  cubic  centi- 
meter of  the  acid  with  4  cubic  centimeters  of  alcohol  and  add  a  drop  or 
two  of  the  bilirubin  solution  to  be  tested.  A  permanent  green  color 
appears,  but  if  stronger  oxidation  is  secured  by  adding  more  of  the  acid 
mixture  the  colors  change  as  in  the  Gmelin  test.  The  reaction  can  be 
well  applied  to  urine. 


CHEMISTRY    OF    THE    LIVER.  325 

FUNCTIONS  AND  BEHAVIOR  OF  BILE. 

The  bile  as  a  whole  has  a  number  of  functions  to  perform  in 
the  body,  some  of  which  have  been  referred  to  in  the  discus- 
sion of  digestive  processes.  It  represents  also  the  avenue  of 
escape  of  a  number  of  by  products  formed  by  the  katabolic 
processes  in  the  liver.  Many  of  these  processes  are  doubtless 
very  complex  and  in  them  a  variety  of  secondary  or  side  re- 
actions occur  which  furnish  matters  of  no  further  use  appar- 
ently in  the  body.  These  are  collected  in  the  gall  bladder  and 
finally  discharged  into  the  small  intestine,  where  escape  from 
the  body  with  the  feces  is  possible  for  the  constituents  having 
no  further  value. 

In  part,  therefore,  the  bile  must  be  regarded  as  an  excretion 
like  the  urine,  but  that  the  parallelism  is  not  complete  is  shown 
by  the  fact  that  a  considerable  absorption  takes  place  from  the 
intestine,  and  products  are  returned  which  find  further  applica- 
tion in  the  organism.  There  is  evidence  to  show  that  this 
portion  returned  from  the  intestine  serves  as  a  cholagogue  to 
stimulate  new  secretion  in  the  liver.  It  is  likely  that  this  free 
secretion  and  flow  of  bile  in  the  liver  is  necessary  for  the  suc- 
cessful completion  of  certain  metabolic  processes  going  on 
there,  so  that  it  may  be  regarded  not  merely  as  an  end  but 
also  as  a  means  toward  an  end. 

The  one  digestive  process  in  which  the  bile  seems  to  play  a 
practically  necessary  part  is  in  the  absorption  of  fats ;  here  its 
action  is  largely  mechanical  as  in  some  way  it  aids  the  passage 
of  the  finely  divided  fat  through  the  intestinal  walls.  The 
general  behavior  of  bile  in  this  respect  may  be  illustrated  by  a 
simple  experiment. 

Ex.  Moisten  two  similar  filter  papers  in  funnels,  one  with  water  and 
the  other  with  bile.  Into  each  filter  pour  some  fatty  oil,  such  as  cotton- 
seed oil  or  olive  oil.  Note  that  while  the  oil  will  not  pass  through  the 
paper  moistened  with  water  a  small  amount  passes  slowly  through  the 
bile-moistened  filter.  Similar  experiments  have  been  made  with  animal 
membranes. 

A  more  important  action  with  fat,  however,  is  shown  in  the 
power  of  bile  to  form  fat  emulsions.    This  is  now  looked  upon 


326  PHYSIOLOGICAL    CHEMISTRY. 

as  the  one  reaction  in  the  intestine  in  which  the  presence  of  bile 
is  actually  practically  essential,  since  the  old  views  of  the  anti- 
septic value  of  the  bile  in  preventing  excessive  intestinal  putre- 
faction have  been  shown  to  be  without  foundation.  In  a  diet 
rich  in  fats  the  emulsifying  behavior  of  the  bile  unquestionably 
comes  into  play  as  a  leading  factor  in  the  final  absorption. 
Experiments  have  been  made  on  animals  in  which  the  flow 
of  the  bile  could  be  diverted  from  the  natural  outlet  into  the 
intestine  by  means  of  a  fistula.  In  such  cases  the  digestion  of 
proteins  and  carbohydrates  seemed  to  suffer  no  change  but  the 
digestion  of  fats  was  always  imperfect  and  a  large  portion 
ultimately  escaped  with  the  feces.  Indirectly  there  ma}-  be 
also  a  loss  in  protein  if  the  fat  in  the  food  in  such  cases 
has  a  rather  low  melting  point  and  is  abundant.  A  fatty  layer 
encloses  portions  of  the  partly  digested  proteins  and  prevents 
access  of  the  digestive  fluids  until  the  lower  stretches  of  the  in- 
testine are  reached,  where  bacterial  changes  soon  get  the  upper 
hand  and  rob  the  protein  of  any  further  food  value.  The 
action  of  bile  in  producing  an  emulsion  with  fatty  oils  may  be 
illustrated  by  experiment.  In  an  earlier  chapter  the  formation 
of  emulsions  by  other  methods  was  shown. 

Ex.  In  a  slightly  warmed  mortar  pour  about  5  cc.  of  bile  and  add  to 
it  one  cc.  of  cottonseed  oil.  Rub  the  two  thoroughly  together  for  several 
minutes,  and  then  add  another  small  portion  of  the  fatty  oil.  An  emul- 
sion forms  slowly,  and  becomes  more  persistent  as  the  working  with  the 
pestle  is  prolonged.  The  amount  of  oil  which  can  be  brought  into  the 
form  of  a  stable  emulsion  with  the  5  cc.  of  bile  depends  largely  on  the 
character  of  the  oil.  The  presence  of  a  small  amount  of  free  fatty  acid 
in  the  cottonseed  oil  aids  materially  in  producing  the  emulsion.  The  weak 
alkalinity  of  the  bile  is  doubtless  an  important  point  here,  as  through 
the  alkali  a  little  soap  is  formed  and  this  may  be  the  chief  factor  in  pro- 
ducing the  emulsion. 

Bile  contains  a  large  amount  of  mucin  as  the  analytical 
table  above  shows.  The  stringy  character  of  the  secretion  is 
due  to  this  substance  which  may  be  recognized  by  several  pre- 
cipitation tests.  The  addition  of  alcohol  in  excess  throws 
down  a  flocculent  mass  which  may  be  separated  by  the  centri- 


CHEMISTRY    OF    THE    LIVER.  32/ 

fuge.  The  addition  of  a  little  acetic  acid  produces  likewise  a 
precipitate.  It  is,  however,  practically  impossible  to  secure 
pure  mucin  in  this  way  as  other  bodies  are  carried  down  with 
the  precipitates  and  their  subsequent  separation  is  difficult. 
The  mucin  of  human  bile  is  said  to  be  nearly  pure,  while  that 
of  other  animals  is  mixed  with  nucleo  albumins. 

BILE   CONCRETIONS.     GALL   STONES. 

Under  conditions  not  well  understood  a  precipitation  of  cer- 
tain constituents  of  the  bile  may  occur  in  the  gall  bladder. 
These  precipitations  take  the  form  of  solid  masses  which  some- 
times grow  to  considerable  size,  by  gradual  surface  additions. 
In  every  case  the  deposited  material  is  built  up  in  layers,  often 
well  defined,  around  some  body  as  a  nucleus.  Three  general 
classes  of  such  calculi  are  recognized.  In  man  balls  of  choles- 
terol, more  or  less  pure,  are  the  most  abundant  while  pigment 
stones  are  also  frequently  found.  These  pigment  stones  con- 
tain essentially  bilirubin  in  combination  with  calcium,  the 
alkali  earth  salts  of  the  pigments  being  insoluble.  The  center 
of  the  cholesterol  stone  may  be  a  nucleus  of  bilirubin  calcium. 
Pigment  stones  are  common  in  the  gall  bladders  of  cattle. 
Finally  we  have  stones  consisting  of  calcium  phosphate  or 
carbonate,  which  however,  are  not  usual  in  man. 

The  following  analyses  made  of  gall-stones  of  very  different 
appearance,  illustrate  the  composition  of  the  cholesterol  stones 
in  man: 

Water    (at  ioo°) 4.60  4.50 

Cholesterol    (and  trace  of  fat) 90.87  90.08 

Bilirubin   (CHCU  extraction) 0.81  )  0.19) 

Biliverdin   (CsHeO  extraction) 2.24  /  ^'^^  1.58  f  ^'77 

Mucin  and  soluble  extractives 0.14  1.53 

Total    ash 0.88  2.72 

Total   P2O5 0.20  i.oo 

These  concretions  frequently  give  rise  to  serious  pathologi- 
cal conditions  and  they  must  then  be  removed  by  surgical  oper- 
ations.    In  addition  to  the  above  constituents  the  stones  con- 


328  PHYSIOLOGICAL    CHEMISTRY. 

tain  small  amounts  of  iron  and  often  traces  of  copper.  But 
the  iron  found  is  far  from  accounting  for  the  amount  which 
must  be  separated  from  the  hematin  in  the  formation  of  bili- 
rubin. In  a  former  chapter  the  preparation  of  cholesterol  from 
gall-stones  was  described,  also  the  general  chemical  behavior 
of  the  substance.  The  character  of  a  stone  is  most  easily  rec- 
ognized by  its  behavior  toward  boiling  alcohol,  in  which 
cholesterol  is  rather  readily  soluble,  to  crystallize  in  large  thin 
plates  on  cooling. 

The  solutions  of  cholesterol  have  a  marked  action  on  polar- 
ized light,  which  property  may  l^e  employed  sometimes  in  the 
identification  and  estimation.  The  specific  rotations  below 
have  been  found. 

Ether  solution    c  =  2  [o]  ^'  =  —  31.12° 

Chloroform   solution    "      2  "  —  37  02° 

"      5  "  -37.81°    . 

"      8  "  -38.63° 

In  feces  a  modified  cholesterol  is  found  which  has  been  called  kopros- 
tcrin  and  also  stcrcorin.  This  new  substance  is  a  reduction  product  with 
the  probable  formula  C2tH4sO  and  is  dextrorotatory,    [a]  ==  +  24°. 

Besides  the  two  principal  pigments  several  derived  substances  have  been 
obtained  from  the  gall-stones.  The  following  have  been  described :  bili- 
fuscin,  biliprasin,  bilihuniin,  bilicyanin.  These  substances  exist  in  small 
amount  and  are  without  practical  importance.  Their  relations  to  the 
others  are  not  clearlv  established. 


CHAPTER     XVIII. 

CHEMISTRY  OF  THE  PANCREAS  AND  OTHER  GLANDS. 
MUSCLE,  BONE.  THE  HAIR  AND  OTHER  TISSUES. 

In  this  chapter  a  number  of  substances  wiH  be  briefly  dis- 
cussed, the  chemical  relations  of  which  in  some  cases  are  un- 
important, or  sometimes,  when  important,  not  well  understood. 
In  regard  to  the  pancreas  it  will  be  recalled  that  in  the  discus- 
sion of  digestive  phenomena  the  behavior  of  active  enzymes  in 
the  liquid  secreted  by  the  organ  was  rather  fully  considered. 
In  the  so-called  pancreatic  juice  the  three  most  important 
enzymes  are  active  in  the  digestion  of  carbohydrates,  fats  and 
proteins,  but  in  addition  to  these  functions  others  must  be 
mentioned. 

THE  PANCREAS. 

The  organ  is  relatively  poor  in  solids,  containing  only  about 
I  GO  parts  per  looo.  The  solid  substance  consists  largely  of 
nucleo  proteids  with  but  comparatively  small  amounts  of  the 
other  protein  bodies.  Besides  producing  the  digestive  en- 
zymes, or  their  zymogens,  the  pancreas  cells  have  an  impor- 
tant function  to  perform  in  connection  with  the  oxidation  of 
sugar  in  the  body.  It  has  long  been  known  that  a  kind  of 
diabetes  results  on  the  extirpation  of  the  pancreas.  Some- 
thing seems  to  be  produced  there  which  is  apparently  essential 
in  the  oxidation  process.  Experiments  with  animals  have 
shown  that  the  oxidation  takes  place  if  even  a'  small  portion 
of  the  organ  is  left.  Of  the  nature  of  the  actiA'e  ferment  here 
or  of  its  mode  of  action  practically  nothing  is  known;  but  it 
has  been  pointed  out  recently  by  several  writers  that  in  this 
sugar  oxidation,  taking  place  in  the  muscles  probably,  two 
things  are  concerned.  The  pancreas  may  furnish  one  of  these 
and  an  enzyme  formed  in  the  muscle  cells  is  the  other.  Cell- 
free   extracts   from  the   organs   taken   separately   have   been 


^^O  PHYSIOLOGICAL    CHEMISTRY. 

found  to  be  practically  inert  toward  sugar,  while  in  presence 
of  a  mixture  of  the  two  extracts  oxidation  follows  readily. 
It  has  been  suggested  that  one  of  these  organs  furnishes  an 
enzyme  which  is  the  cataly::cr  for  the  other,  and  attempts  have 
been  recently  made  to  produce  the  pancreas  enzyme  on  a  large 
scale  for  use  therapeutically. 

Autolysis.  The  pancreas  readily  undergoes  autolytic  di- 
gestion under  the  aseptic  treatment  or  when  preserved  by 
toluene.  A  large  number  of  products  may  be  separated  from 
the  altered  mass,  which  in  a  general  way  resemble  those  pro- 
duced in  the  liver,  as  already  referred  to.  Ammonia,  leucine, 
tyrosine,  aspartic  acid,  glutaminic  acid  and  the  hexone  bases 
have  been  recogiiized ;  also,  the  somewhat  unusual  oxyphenyl- 
ethylamine.  HO  •  QjH^  •  CH2  •  CHoNH,,  which  may  be  de- 
rived from  tyrosine  by  splitting  off  of  COo. 

THE  SUPRARENAL  BODIES. 

A  soluble  substance  contained  in  the  capsules,  because  of 
its  important  property  of  raising  the  blood  pressure,  has  at- 
tracted a  great  deal  of  attention  in  the  last  ten  years.  This 
soluble  substance  was  first  recognized  as  a  chromogen  which, 
on  account  of  its  oxygen-absorbing  power,  was  assumed  to  be 
related  to  pyrocatechol.  An  aqueous  extract  of  the  capsules 
becomes  dark  on  exposure  to  the  air  and  produces  a  dark 
green  color  when  treated  with  ferric  chloride.  It  also  reduces 
Fehling's  solution  strongly  and  shows  the  same  behavior 
toward  other  metallic  salts.  The  oxygen-absorbing  power  of 
the  extract  had  been  known  about  thirty  years  before  the  im- 
portant relation  to  blood  pressure  was  discovered.  It  was 
soon  found  that  the  two  properties  seem  to  reside  in  the  same 
constituent  of  the  extract,  since  the  destruction  of  one  is  fol- 
lowed by  the  disappearance  of  the  other.  Numerous  investi- 
gations have  been  carried  out  on  the  isolation  of  the  active 
principle,  especially  by  Abel,  v.  Fiirth  and  Takamine,  who 
have  given  the  names  epinephriii,  snprarcn'ui  and  adrenalin 


THE    SUPRARENAL    BODIES.  331 

respectively  to  active  extracts  which  they  have  separated  by 
different  processes.  Some  idea  of  the  nature  of  the  substance 
may  be  obtained  from  considering  a  method  given  by  Taka- 
mine  for  separating  it. 

The  minced  capsules  are  extracted  by  weakly  acidulated  water  in  an 
atmosphere  of  carbon  dioxide  to  prevent  oxidation.  The  temperature  of 
the  extraction  is  at  first  50°-6o°  and  finally  90°-95°  to  coagulate  proteins. 
The  extract  is  concentrated  in  vacuo  and  precipitated  with  strong  alcohol ; 
the  filtrate  is  concentrated — the  alcohol  distilled  off — in  vacuo,  and  to  the 
aqueous  residue  ammonia  is  added.  This  produces  a  precipitate  of  the 
active  principle  in  crude  form,  which  crystallizes  in  time.  The  precipi- 
tate is  redissolved  with  a  little  acid  in  alcohol,  and  certain  impurities  are 
thrown  out  by  addition  of  ether.  The  filtrate  is  concentrated  in  vacuo 
again  and  a  new  precipitation  effected  by  ammonia.  By  repeating  this 
treatment  several  times  a  much  purer  product  is  obtained. 

A  Hght  yehowish  crystalHne  substance  is  finaUy  secured 
which  has  been  several  times  analyzed,  but  not  with  abso- 
lutely concordant  results.  The  formula  C10H15NO3  has  been 
proposed  by  Takamine,  but  may  not  quite  represent  the  com- 
position. The  adrenalin  is  slightly  soluble  in  cold  water  and 
much  better  in  hot.  Weak  acids  and  alkali  hydroxides  aid 
solution,  but  ammonia  produces  a  precipitate.  Salts  are 
formed  with  the  common  acids,  and  of  these  the  hydrochloride 
is  used  in  medicine  already  extensively,  on  account  of  its  blood 
pressure-raising  property.  This  salt  seems  to  contain  one 
molecule  of  HCl  with  one  of  the  active  principle. 

On  cleavage  with  alkali  adrenalin  furnishes  a  number  of 
products  which  give  some  idea  of  its  possible  composition. 
Pyrrol,  skatol  and  protocatechuic  acid  have  been  obtained; 
three  hydroxyl  groups  seem  to  be  present  and  one  methyl- 
amine  group,  v.  Fiirth  suggests  a  cyclic  formula  with  one 
CH2  less  than  the  formula  given  above  with  a  possible  con- 
stitution [(CH3)NC2HOH]C6He(OH)2.  Other  groupings 
for  the  empirical  formula  C9H13NO3  have  also  been  given. 
Recently  the  specific  rotation  of  the  acetate  has  been  found. 
This  is,  at  23.5°,  [a] ^  =  —  43°. 

The  other  constituents  of  the  suprarenal  capsules  have  no 
importance  at  the  present  time  that  can  be  clearly  defined,  but 


332  PHYSIOLOGICAL    CHEMISTRY, 

as  is  well  known,  complete  removal  of  the  lx»dies  is  usually 
attended  with  fatal  results,  and  Addison's  disease  is  associated 
with  certain  pathological  conditions  in  the  organs.  Lecithin 
bodies  and  a  glucose  furnishing  complex  are  present  in  small 
amount,  as  well  as  the  mass  of  protein  substance  which  has 
not  yet  been  fully  investigated. 

THE  THYROID  GLAND. 

The  relation  of  this  gland  to  certain  pathological  conditions 
which  sometimes  appear  in  man  and  which  may  be  induced 
in  animals  has  been  a  subject  of  study  for  many  years.  At- 
tempts to  isolate  the  active  principle  or  principles  on  which 
the  functions  of  the  gland  depend  have  been  in  a  measure  suc- 
cessful. In  the  course  of  investigations  a  number  of  basic 
bodies  have  been  separated,  but  these  may  have  no  connection 
with  the  obser\-ed  physiological  behavior. 

From  the  investigations  of  Oswald,  who  has  made  the  full- 
est contributions  to  the  literature,  there  are  two  peculiar  pro- 
tein bodies  present,  one  of  which  is  a  globulin  and  the  other 
a  nucleo  proteid.  To  the  first  he  has  given  the  name  thyrco- 
glohuUn:  this  exists  frequently  combined  with  iodine,  and  it 
is  the  latter  complex  which  is  theoretically  and  practically 
important.  It  has  been  called  iodothyrcoglohiilin  and  appears 
to  be  found  only  in  those  glands  which  contain  colloid,  and 
the  amount  of  iodine  present  is  proportional  to  the  amount  of 
colloid.  The  normal  gland  weighs  usually  30-45  grams,  in 
which  the  thyreoglobulin  fraction  is  in  the  mean  about  ten 
per  cent.  The  amount  of  iodine  is  usually  less  than  one-tenth 
of  one  per  cent  of  the  whole.  In  case  of  enlarged  glands — 
goitre — the  whole  organ  may  weigh  up  to  several  hundred 
grams.  If  the  goitre  is  rich  in  colloid  the  iodine  appears  to 
be  absolutely,  but  not  relatively,  increased.  In  the  thyreo- 
globulin from  a  normal  gland  over  0.3  per  cent  of  iodine  has 
been  found,  while  in  the  preparation  from  colloid  goitres  the 
amount  in  the  mean  is  0.06-0.07  per  cent. 

By  treatment  with  acids  the  gland,  or  the  thyreoglobulin 


THE    REPRODUCTIVE    GLANDS.  333 

from  it,  undergoes  a  cleavage  in  which  a  residue  rich  in  iodine 
remains.  The  organic  iodine  compound  so  obtained  which 
may  be  the  true  active  principle  is  called  iodothyrin  or  thyro- 
iodine.  In  earlier  experiinents  Baumann,  the  discoverer  of 
this  compound,  found  an  iodine  content  of  about  9  per  cent, 
but  Oswald,  starting  with  pure  iodothyreoglobulin  which  was 
secured  by  a  salting-out  process  with  ammonium  sulphate, 
obtained  finally  iodothyrin  with  over  14  per  cent  of  combined 
iodine.  This  iodothyrin  is  not  a  protein  substance ;  the  analy- 
ses of  different  preparations  are  not  in  very  good  accord,  from 
which  it  appears  that  the  pure  substance  has  not  yet  been 
actually  secured.  The  crude  product  at  present  known  has 
been  used  in  medicine  and  attempts  have  been  made  to  dupli- 
cate or  replace  it  by  other  iodine  compounds. 

The  administration  of  iodine  salts  increases  the  iodine  con- 
tent of  the  thyreoglobulin  in  animals.  It  has  also  been  shown 
that  extracts  from  the  gland,  or  the  iodothyrin  when  adminis- 
tered hypodermically,  counteract  the  bad  effects  of  the  re- 
moval of  the  gland  or  the  effects  of  its  degeneration.  The 
therapeutic  uses  depend  on  these  observations. 

The  other  extractive  bodies  found  in  the  thyroid  are  not 
important.  Several  of  the  purine  derivatives  seem  to  be  pres- 
ent ;  also  lactic  and  succinic  acids  and  amino  acids,  probably  as 
degeneration  products.  The  thymus  gland  of  the  calf  is  said 
to  resemble  the  thyroid  in  containing  a  little  iodothyrin. 

THE  REPRODUCTIVE  GLANDS. 

Of  the  chemical  composition  of  the  testicles  and  their  secre- 
tion not  much  can  be  said.  The  testicles  contain  several  pro- 
teins and  extractives,  but  their  investigation  has  been  ex- 
tremely limited.  The  most  complete  examinations  of  the 
spermatic  fluid  are  probably  those  reported  by  Slowtzoff,  from 
whose  work  the  following  figures  are  taken.  The  specific 
gravity  of  the  fluid  varies  from  1.02  to  1.04;  the  reaction  is 
alkaline  and  as  measured  by  the  aid  of  rosolic  acid  corresponds 
to  0.15  per  cent  sodium  hydroxide.  As  a  mean  of  five  analy- 
ses the  following  results  may  be  given : 


334  PHYSIOLOGICAL    CHEMISTRY. 

Spermatic  Fluid. 

Specific  gravity   1.0299 

Water  90.32  per  cent. 

Dry  substance   9.68 

Salts    0.90 

Proteins   2.09 

Ether   extract    0.17 

Water  and  alcohol  extracts    6. 11 

The  tables  below  show  the  calculations  for  dry  substance 
and  tlie  character  of  the  ash : 

For   Dry   Substance.  Ash. 

Organic    90.81  per  cent.        NaCl    29.05  per  cent. 

Inorganic   9.19        "  KCl    3.12        " 

Proteins    24.48        "  Sd    11.72        " 

Ether  extract  ...  2.15        "  CaO   22.40        " 

Water    and    alco-  P^Os    28.79 

hoi  extract    . . .  59.36        " 

The  ash  is  peculiar  in  containing  a  large  amount  of  sodium 
chloride  and  calcium  phosphate.  The  phosphoric  acid  is  pres- 
ent in  larger  amount  than  corresponds  to  the  nuclein  sub- 
stances. 

The  proteins  are  made  up  approximately  as  follows : 

Albumins    68.5 

Albumose-like   bodies    21.6 

Nucleins   9.9 

A  characteristic  basic  body  known  as  spermine  is  present 
in  small  amount.  The  empirical  formula  CoHgN  has  been 
given  to  it.  This  substance  forms  a  combination  with  phos- 
phoric acid  which  sometimes  separates  in  cr^'stalline  form  on 
evaporation  of  the  fluid.  The  characteristic  odor  of  the  dis- 
charged secretion  is  said  to  be  due  to  partial  decomposition  of 
the  base. 

The  spermatozoa  are  relatively  stable  bodies  and  resist  the 
action  of  chemical  reagents  to  a  remarkable  degree.  The 
heads  of  spermatozoa  consist  largely  of  nuclein  compounds 
while  the  tails  contain  other  proteins,  cholesterol  fat  and 
lecithin.  The  ash  content  of  the  whole  is  relatively  high  and 
is  rich  in  potassium  phosphate. 


BRAIN    AND    NERVE    SUBSTANCES.  335 

BRAIN  AND  NERVE  SUBSTANCES.      CEREBRO- 
SPINAL LIQUID. 

These  tissues  contain  several  peculiar  compounds  of  which 
our  knowledge  is  limited,  largely  because  of  the  great  difficulty 
in  separation.  The  solid  matter  of  the  brain  contains  globu- 
lins, nucleo  proteids,  cholesterol,  lecithin,  fatty  bodies  and 
complex  compounds  not  found  elsewhere.  Various  soluble 
extractives,  somewhat  similar  to  those  from  muscular  tissue, 
are  also  present. 

Protagon.  This  is  an  important  constituent  of  the  white 
substance  of  the  brain,  which  has  this  elementary  composi- 
tion, according  to  Gamgee :  C  66.4,  H  1.07,  N  2.4,  P  1.07. 
But  as  other  writers  report  rather  widely  different  figures  it 
is  likely  that  the  pure  substance  has  not  yet  been  isolated.  As 
extracted  by  means  of  85  per  cent  alcohol  at  45°  from  the 
minced  brain,  and  purified  by  crystallization  and  washing  with 
ether,  it  is  obtained  as  a  white  powder  practically  insoluble  in 
cold  ether  or  alcohol  and  not  properly  soluble  in  water.  With 
much  water  it  finally  yields  a  gelatinous  liquid,  which  suffers 
decomposition  readily. 

When  warmed  with  dilute  alkalies  several  cleavage  prod- 
ucts are  obtained  to  which  the  names  cerehrin,  kerasin,  cerc- 
bron  and  encephalin  have  been  given.  These  bodies  are  free 
from  phosphorus.  On  further  cleavage  with  dilute  acid  they 
yield  a  reducing  sugar,  probably  galactose.  No  satisfactory 
analyses  of  these  products  have  as  yet  been  obtained.  It  is 
possible  that  they  exist  as  glucosides. 

In  the  white  substance  of  the  spinal  marrow  protagon  is 
abundant.  In  degeneration  changes  in  the  tissues  of  the  ner- 
vous system  it  is  probably  this  compound  which  suffers  the 
greatest  alteration,  with  the  production  of  neurine  or  choline 
with  marked  toxic  properties.  It  is  likely  that  the  neurine 
comes  from  a  lecithin  body  as  one  of  the  groups  in  the  prota- 
gon complex. 

Cerebro-spinal  Liquid.     This  is  a  thin,  watery  liquid  of 


336  I'llVSIOLOC.ICAL    CHEMISTRY. 

which  only  a  few  partial  analyses  have  been  recorded.  Its 
general  character  is  shown  by  these  tigiires  recently  given  by 
Zdorek : 

1000  parts  by  weight  contain 

Dry   substance    1045 

Organic   2.09 

Inorganic    8.36 

Proteins    0.77 

Chlorine    4.24 

Sodium    oxide    4.29 

The  organic  substance  includes  traces  of  fats,  lecithin, 
cholesterol  and,  pathologically,  choline  or  neurine.  Common 
salt,  however,  is  the  main  solid  substance  in  solution. 

MUSCLE  AND  ITS  EXTRACTIVES. 

A  large  part  of  the  solid  portion  of  the  body  is  made  up  of 
muscular  tissue.  A  knowledge  of  the  composition  of  this 
tissue  is  of  the  highest  importance,  especially  since  some  of 
the  fundamental  chemical  reactions  of  the  animal  organism 
take  place  within  the  cells  of  the  muscles.  Fortunately  we 
have  fairly  satisfactory  information  on  some  of  the  points  of 
interest  here,  as  numerous  analyses  have  been  made  of  the 
muscles  and  of  the  liquid  which  may  be  extracted  in  various 
ways  from  them. 

The  dry  part  of  the  muscle  is  made  up  largely  of  proteins 
of  which  several  are  present ;  in  the  muscle  plasma  there  are 
at  least  five  according  to  Halliburton.  In  addition  to  these 
bodies  there  are  a  number  of  so-called  extractives  which  play 
an  important  part. 

GENERAL   COMPOSITION   OF   MUSCLE. 

The  following  figures  represent  approximately  the  average 
composition  of  the  fresh  muscle  dissected  free  from  visible 
fat. 

Water    76  per  cent. 

Solids     24        " 


MUSCLE    AND    EXTRACTIVES.  337 

Proteins    (true) i7-6 

Collagen    substance    3-0 

Fat,    interstitial    i-5 

Flesh  bases   0.2 

N-free   extractives    0.4 

Salts    1-3 

The  Muscle  Proteins.  It  is  not  possible  to  give  a  per- 
fectly clear  account  of  all  these  bodies  at  the  present  time,  as 
the  products  obtained  by  different  investigators  vary  with  the 
details  of  the  extraction  methods  employed..  The  more  im- 
portant constituents  commonly  recognized  are  indicated  in  the 
following  paragraphs.  By  washing  out  the  blood  from  living 
muscle  by  physiological  salt  solution  (transfusion),  dissecting 
it,  grinding  it  to  a  pulp  and  pressing  very  strongly  a  clear 
yellowish  liquid  is  obtained  which  is  called  muscle  plasma. 
The  ordinary  dead  muscle  treated  in  the  same  manner  yields 
a  different  liquid  which  may  be  called  muscle  serum.  The 
plasma  has  an  alkaline  reaction  and  is  distinguished  by  the 
property  of  spontaneous  coagulation. 

The  term  myosin  was  formerly  applied  to  the  solidified  or 
coagulated  body  as  a  whole,  but  experiment  shows  that  two 
things  at  least  are  here  present.  One  of  these  is  called  dius- 
cnlin,  or  by  some  authors,  myosin  proper,  while  the  other  prod- 
uct is  known  as  myogen.  The  musculin,  or  myosin,  coagu- 
lates at  about  47°,  while  for  myogen  the  coagulating  tempera- 
ture is  about  56°. 

The  two  substances,  musculin  and  myogen,  differ  also  in 
their  precipitation  properties.  The  first  is  precipitated  from 
solution  by  adding  ammonium  sulphate  to  make  up  28  per 
cent;  from  the  filtrate  the  myogen  may  be  thrown  down  by 
adding  the  sulphate  to  saturation,  and  is  found  to  make  up 
about  80  per  cent  of  the  plasma  protein. 

The  serum  left  after  the  formation  of  the  plasma  coagulum 
usually  contains  a  little  soluble  albumin.  This  may  be  nor- 
mal to  the  muscle  substance, -or  it  may  be  due  to  the  blood  not 
perfectly  removed  by  the  preliminary  washing.  At  any  rate 
the  plasma  consists  essentially  of  the  two  myosin  bodies. 
23 


T,^S  PHYSIOLOGICAL    CHEMISTRY. 

After  separation  of  the  plasma  what  may  l)e  called  the 
stroma  remains.  This  is  mainly  alhiiminous.  but  its  exact 
nature  is  not  known.  The  sarcolemma  portion  of  the  muscle 
fiber,  which  by  weight  makes  up  but  a  small  part  of  the  whole, 
appears  to  belong  to  the  albumoid  group  of  proteins,  resem- 
bling elastin.  It  has  been  shown  in  an  earlier  chapter  that 
from  ordinary  dead  muscle,  as  represented  by  lean  meat,  a  con- 
siderable amount  of  "  myosin  "  may  be  separated  by  extract- 
ing with  a  weak  solution  of  ammonium  chloride.  What  re- 
mains does  not  agree  fully  with  the  stroma  left  on  pressing 
out  the  plasma  of  the  fresh  muscle,  but  contains  approxi- 
mately the  same  substances.  By  this  method  of  separation 
the  insoluble  stroma  portion  is  much  larger  than  the  soluble 
or  "  myosin  "  portion.  The  latter  may  amount  to  7  or  8  per 
cent  of  the  weight  of  the  muscle  in  the  mean. 

Collagen.  As  given  in  the  above  table  this  refers  to  the 
binding  substance  holding  the  muscle  fibers  together  and  in- 
cludes the  sarcolemma.  It  is  insoluble  in  cold  water,  but 
swells  and  disintegrates  finally  in  boiling  water. 

Fat.  After  removing  all  visible  fat  from  the  dissected 
muscle,  analyses  still  show  a  small  amount  remaining.  This 
must  therefore  be  associated  with  the  minute  structure  of  the 
fibrils. 

Flesh  Bases.  A  number  of  ver\'  remarkable  substances 
are  included  here.  They  are  sometimes  described  as  the 
nitrogenous  extractives.  The  most  abundant  of  these  bodies 
is  creatine  or  methylguanidine  acetic  acid ;  some  of  the  purine 
bases  are  also  present.  A  brief  description  of  these  sub- 
stances may  be  given. 

Creatine,  C4H9N3O2.  may  be  represented  structurally  by 
the  formula 


H— N=C< 


^  CH,  .  COOH 


It  is  found  in  all  muscles  and  is  a  product  of  metabolism. 
Being  readily  soluble  in  w^arm  water  and  in  about  75  parts 


MUSCLE    AND    EXTRACTIVES.  339 

of  water  at  the  ordinary  temperature  its  extraction  from  mus- 
cle is  easy.  When  the  solution  is  boiled  with  dilute  hydro- 
chloric acid  a  molecule  of  water  is  split  off  and  the  anhydride 
creatinine  is  left.  This  is  a  normal  urinary  constituent  and 
will  be  described  later.  When  boiled  with  alkali  solution, 
especially  baryta  water,  creatine  undergoes  a  complete  cleav- 
age into  urea  and  sarcosine,  which  relation  is  an  interesting 
one  and  has  suggested  the  possible  derivation  of  the  urinary 
urea.  Creatine  may  be  readily  crystallized  from  water  solu- 
tion. It  was  formerly  made  for  experiment  directly  from 
meat.  It  is  best  secured  from  certain  crystalline  residues  oc- 
curring as  by-products  in  the  manufacture  of  "  beef  extract," 
referred  to  below. 

Carnine.  The  amount  of  this  in  muscle  is  very  small, 
but  it  may  be  recognized  in  beef  extract.  It  bears  some  rela- 
tion in  structure  to  hypoxanthine  and  has  been  given  the  for- 
mula C.HgN^Og. 

The  Xanthine  Bodies.  These  constitute  a  peculiar 
group  of  great  importance  because  of  their  relation  to  uric 
acid.  Traces  of  several  of  them  have  been  recognized  in  the 
muscular  juices;  in  a  later  chapter  the  structure  and  proper- 
ties of  the  substances  will  be  discussed  in  connection  with  uric 
acid.     Traces  of  urea  are  also  found  in  the  muscles. 

The  Nitrogen-Free  Extractives.  The  muscular  juices 
hold  dissolved  a  number  of  compounds  which  contain  no  ni- 
trogen, some  of  which  are  very  important.  The  chief  of 
these  are  glycogen,  inosite,  glucose  and  lactic  acid. 

Glycogen.  The  chemical  relations  of  glycogen  have  been 
discussed  already  in  earlier  chapters.  The  glycogen  as  found 
in  the  muscles  comes  from  the  liver,  being  transported  there 
by  the  blood,  and  in  part  is  probably  formed  in  the  muscles 
by  the  same  kind  of  an  enzymic  action  which  leads  to  its  syn- 
thesis in  the  liver.  The  liver  is  capable  of  storing  up  a  large 
weight  of  the  reserve  substance  in  a  small  space.  The  amount 
stored  in  an  equal  weight  of  muscle  is  small,  but  taking  the 
muscles  of  the  body  as  a  whole  the  glycogen  content  is  con- 
siderable, reaching  a  hundred  grams  or  more. 


340  PHYSIOLOGICAL    CHEMISTRY. 

It  is  probably  through  this  glycogen  that  the  muscle  is  capa- 
ble of  doing  its  work.  Through  enzymic  hydration  the  gly- 
cogen becomes  sugar,  possibly  maltose  and  then  glucose,  and 
the  potential  energy  of  this  is  liberated  by  oxidation  to  water 
and  carbon  dioxide  ultimately.  The  oxidation  may  not  be 
direct ;  in  all  probability  there  are  several  transformation  prod- 
ucts before  the  final  stages  are  reached.  But  the  energy  trans- 
formation is  the  same  whatever  the  intermediate  steps  may  be. 
The  importance  of  the  glycogen  and  related  bodies  in  this 
direction  will  be  pointed  out  in  a  following  chapter.  It  may 
be  recalled  that  in  these  oxidation  processes,  where  sugar  is 
concerned,  a  nmscle  enzyme  and  a  pancreas  enzyme  seem  to  be 
both  necessary. 

While  glycogen  in  the  muscles  must  come  mostly  from 
sugars,  either  directly  or  through  the  liver,  there  is  also  some 
evidence  that  it  may  come  in  part  from  other  substances,  espe- 
cially from  proteins.  Animal  experiments  have  shown  ap- 
parently a  storing  of  glycogen  from  a  protein  diet  after  pre- 
vious starvation  had  exhausted  the  reserve  in  store.  In  the 
breaking  down  of  some  proteins  it  has  been  shown  that  certain 
carbohydrate  groups  are  liberated ;  it  is  doubtless  these  which 
undergo  synthesis  to  form  at  least  part  of  the  glycogen,  and 
from  this  standpoint  the  behavior  of  protein  as  a  glycogen 
factor  is  not  so  hard  to  understand. 

The  glycogen  content  of  the  muscles  of  different  animals 
is  variable;  in  the  flesh  of  the  horse  it  is  relatively  high, 
amounting  often  to  over  i  per  cent.  As  the  muscle  glycogen 
is  not  altered  rapidly  in  the  dead  organ,  as  is  the  liver  glyco- 
gen, the  presence  of  the  substance  in  horse-flesh  sausage  may 
be  quite  readily  recognized.  Methods  have  been  devised  for 
the  identification  of  horse-flesh,  sold  for  food,  based  on  these 
facts.  Glycogen  may  be  extracted  from  the  muscles  by  the 
general  method  given  for  the  liver  in  an  earlier  chapter;  the 
chemical  and  optical  properties  may  be  used  for  the  final  iden- 
tification. 

Inosite.       This-    substance     has     the    empirical     formula 


MUSCLE    AND    EXTRACTIVES.  34 1 

CfiHioOg  +  H2O  and  was  long  spoken  of  as  muscle  sugar. 
It  is  not  a  true  carbohydrate,  however,  but  an  aromatic  prod- 
uct CfjHf;(OH)f;.  that  is,  hexahydroxybenzene.  The  amount 
found  in  muscle  is  very  small  and  how  it  is  derived  is  not 
known ;  but  it  is  not  peculiar  to  these  tissues,  as  it  occurs  in 
other  organs  of  the  body  and  also  in  many  vegetable  sub- 
stances. It  may  be  extracted  from  muscles  without  much 
trouble  and  when  pure  is  found  to  be  a  white  crystalline  pow- 
der melting  at  about  220°.  It  is  very  soluble  in  water  to 
which  a  sweetish  taste  is  given,  and  in  presence  of  alkali  is 
not  a  reducing  agent  for  metallic  solutions.  Although  the 
usual  structural  formula  does  not  show  an  asymmetric  car- 
bon atom  the  substance  is  optically  active  and  exhibits  a  strong 
rotation,  both  right  and  left  forms  being  known. 

Glucose.  From  what  was  said  above  about  the  transfor- 
mation of  glycogen  it  is  not  surprising  that  a  small  amount  of 
sugar  should  be  found  in  the  muscles ;  both  maltose  and  glu- 
cose have  been  detected. 

Lactic  Acid.  Several  forms  of  this  acid  are  known,  but 
that  occurring  in  the  muscle  is  the  dextrorotatory  paralactic 
or  sarcolactic  acid,  CgHeOg.  It  is  one  of  the  a-hydroxypro- 
pionic  acids.  There  has  been  much  speculation  as  to  the 
source  of  this  acid  in  the  body,  but  it  seems  most  rational  to 
regard  it  as  derived  from  the  glycogen  or  sugar  by  a  com- 
paratively simple  cleavage.  It  is  also  possible  that  in  the 
katabolic  reactions  of  proteins  lactic  acid  may  result  from  a 
splitting  of  the  carbohydrate  group.  The  acid  is  not  very 
readily  detected  in  the  living  muscle  because  it  is  probably 
oxidized  or  removed  too  rapidly  by  the  fluid  circulation.  In 
the  dead  muscle,  however,  it  may  accumulate  to  the  extent  of 
half  a  per  cent  or  more.  The  living  muscle  shows  a  neutral 
or  slightly  alkaline  reaction,  while  in  the  dead  muscle  the 
increase  of  lactic  acid  changes  the  reaction. 

The  lactic  acid  of  the  muscle  probably  results  from  an  en- 
zymic  cleavage.  In  the  aseptic  autolysis  of  liver  paralactic 
acid  has  been  recognized  among  the  products,  and  this  fact 


342  PHYSIOLOGICAL    CHEMISTRY. 

shows,  at  least,  the  possibihty  of  such  a  formation.  The 
amount  of  lactic  acid  formed  in  the  muscle  seems  to  be  great- 
est during-  working  periods,  which  is  true  also  of  the  final 
products  of  katabolism.  The  acid  may  simply  represent  a 
stage  in  the  gradual  breaking  down,  whether  we  consider  a 
carbohydrate  or  protein  as  the  parent  substance.  We  should 
expect  therefore  an  increase  in  the  muscle  acid  if  the  oxida- 
tion processes  of  the  body  are  hindered  or  retarded,  while  at 
the  same  time  protein  or  sugar  decomposition  is  increased. 
or,  at  any  rate,  not  diminished.  In  the  dead  muscle  the  en- 
zymic  formation  of  lactic  acid  doubtless  continues  long  after 
the  oxidation  reaction  ceases,  and  this  is  probably  the  main 
reason  for  the  ready  detection  in  the  muscle  after  death. 

The  pure  acid  occurs  as  a  thickish  liquid  miscible  with 
water.  It  forms  salts  which  are  mostly  readily  soluble.  The 
zinc  and  calcium  salts  crystallize  well  and  are  hence  prepared 
for  identification.  The  pure  liquid  shows  a  right  hand  optical 
rotation,  with  [a] ^  =  about  3".  The  result  is  not  constant 
because  of  the  difficulty  of  preparing  concentrated  solutions 
free  from  anhydride  or  lactide.  The  rotation  of  the  salts,  on 
the  contrary,  is  to  the  left. 

The  Inorganic  Salts.  Although  making  up  not  much 
over  I  per  cent  of  the  weight  of  the  moist  muscle,  these  salts 
are  extremely  important.  Of  dry  substance  the  salts  consti- 
tute 5  per  cent  or  more.  The  salts  are  usually  estimated  from 
the  ash  left  in  burning  the  muscle;  this  gives  of  course  no 
correct  idea  of  how  they  are  combined  in  the  living  muscle, 
but  is  the  only  method  available.  In  the  living  muscle  many 
of  the  inorganic  elements  are  doubtless  in  chemical  union  with 
proteins  or  other  organic  groups,  while  in  the  derived  ash  we 
have  chlorides,  phosphates,  sulphates  or  carbonates.  A  car- 
bonate is  probably  formed  during  the  combustion  of  organic 
acids  and  corresponds  to  no  simple  preexisting  compound. 
Phosphorus  and  sulphur  of  proteins  furnish  phosphates  and 
sulphates.  The  analyses  of  ash  made  disclose  very  different 
results,  but  mean  values  may  be  given  to  show  the  general 


MUSCLE    AND    EXTRACTIVES.  343 

approximate  composition.     In  the  calculation  carbonic  acid  is 
not  considered.     The  table  below  is  from  the  Konig  collection. 

K2O 3704 

Na20    10. 14 

CaO   2.42 

MgO    323 

FCaOs    0.44 

P2O5    41.20 

SO3 0.98 

CI     4.66 

SiO-  0.69 

From  the  table  it  appears  that  potassium  phosphate  is  the 
most  abundant  substance  in  the  ash.  Much  of  this  doubtless 
preexists  in  the  muscle  juices,  while  a  small  portion  is  of 
oxidation  origin.  The  small  sulphate  content  is  probably  due 
to  protein  sulphur  fully  oxidized  in  the  combustion.  In  the 
past  too  little  attention  has  been  given  to  the  mineral  constitu- 
ents of  the  body,  it  being  commonly  assumed  that  they  repre- 
sent "  waste  "  or  "  ash  "  only.  But  the  newer  applications  of 
chemistry,  especially  physical  chemistry,  to  physiology  have 
disclosed  the  fact  that  the  inorganic  salts  are  especially  con- 
cerned in  the  proper  maintenance  of  many  of  the  body  func- 
tions. The  balanced  osmotic  pressure  of  the  body  fluids  is 
largely  a  function  of  the  salt  content,  and  variations  here  are 
of  great  importance.  The  mineral  salts  are  the  carriers  of 
electric  charges  in  the  body  and  as  such  seem  to  have  im- 
portant duties  to  perform. 

EXTRACT    OF   MEAT. 

By  boiling  lean  meat  with  water  the  soluble  constituents  are 
dissolved,  producing  an  extract.  When  this  is  concentrated 
to  a  paste  the  article  known  commercially  as  "  Extract  of 
Meat "  results.  The  article  was  first  made  in  quantity  in 
South  America  to  utilize  the  carcasses  of  cattle  slaughtered 
for  the  hides,  but  later  the  manufacture  was  introduced  else- 
where, and  generally  to  utilize  certain  waste  or  by  products  in 
the  meat  industries.     At  first  the  extract  was  assumed  to  pos- 


344  PHYSIOLOGICAL    CHEMISTRY. 

sess  food  value  in  a  high  degree,  but  after  a  time,  as  the  chem- 
istry of  the  proteins  and  their  derivatives  became  better  under- 
stood, this  notion  was  gra(kially  abandoned.  Lean  meat,  mus- 
cle, is  employed  practically  in  the  process ;  hence  little  or  no 
fat  can  be  present.  At  the  boiling  temperature  nearly  the 
whole  of  the  proteins  are  coagulated  and  are  filtered  out.  A 
little  gelatin  remains,  but  the  food  value  of  this  is  of  minor 
importance.  Unless  the  boiling  is  greatly  prolonged  the  ex- 
tract must  therefore  contain  essentially  the  meat  bases  and 
other  extractives  referred  to  above,  and  the  actual  nutritive 
value  of  these  is  low,  in  the  case  of  the  bases  being  nil.  On 
prolonged  boiling,  however,  a  small  portion  of  the  original 
protein  seems  to  pass  over  into  the  soluble  form  of  albumose, 
which  is  therefore  found  in  some  extracts.  Finally,  the  phos- 
phates and  other  inorganic  salts,  being  largely  soluble,  pass 
into  the  extract  and  constitute  a  considerable  part  of  the  fin- 
ished pasty  product. 

In  this  country  "  extract  "  is  made  by  concentrating  the 
broth  resulting  from  the  boiling  of  beef  as  a  step  in  the  can- 
ning process.  Large  quantities  of  meat  being  boiled  in  the 
same  water,  it  becomes  rich  in  the  "  extractives  "  and  is  finally 
boiled  down  to  the  usual  pasty  condition.  Before  the  concen- 
tration is  complete  the  liquid  is  filtered  and  skimmed  and 
therefore  leaves  a  residue  free  from  fat  or  fiber.  Roughly 
speaking  the  paste  extract  has  about  this  composition  : 

Water 20 

Salts   20 

Organic  substances    60 

Numerous  analyses  have  been  made  of  some  of  the  commer- 
cial extracts,  but  the  methods  employed  have  not  always  been 
delicate  enough  to  furnish  trustworthy  information.  This  is 
esi^ecially  true  as  regards  the  amounts  of  so-called  peptone 
and  albumose  present,  for  which  the  definitions  have  not  been 
fairly  uniform  until  comparatively  recently.  The  recognized 
relations  of  these  substances  are  explained  in  the  chapter  on 
protein   compounds.     Analyses   made   by   the  older   methods 


MUSCLE    AND    EXTRACTIVES.  345 

were  generally  reported  as  showing  more  or  less  "  peptone  "' 
when,  according  to  the  present  views,  "  albumose  "  is  meant. 
The  following  figures  may  be  taken  as  representing  approxi- 
mately the  average  composition  of  typical  samples  of  Ameri- 
can meat  extract : 

Water    ' 20.0 

Inorganic  salts    (ash) 22.5 

Albumose    (and   gelatin) ._ 16.5 

Flesh  bases,  etc 26.4 

N-free    extractives    14.6 

According  to  these  results  the  food  value  of  the  extract 
would  be  measured  by  the  nitrogen-free  extractives  and  the 
albumose  and  gelatin  fractions.  In  some  kinds  of  extract  the 
flesh  bases  and  related  bodies  are  much  higher  than  here  given, 
with  corresponding  diminution  in  the  other  organic  constitu- 
ents. The  real  value  of  these  extracts  lies  mainly  in  other 
directions,  however.  They  contain  the  flavoring  and  stimu- 
lating portions  of  the  meat,  and  should  not  be  considered  so 
much  as  foods  as  additions  to  foods.  Added  to  vegetables 
they  impart  an  agreeable  taste  and  doubtless  serve  a  very 
useful  purpose  in  stimulating  appetite  for  substances  not  in 
themselves  possessing  much  flavor.  In  their  action  the  basic 
and  similar  substances  in  the  meat  extracts  may  be  perhaps 
fairly  compared  with  the  alkaloids  in  tea  and  coffee,  which, 
experience  shows,  have  a  real  value.  Large  amounts  of  the 
extracts  cannot  be  used,  however,  as  foods,  because  of  the 
presence  of  the  large  percentages  of  alkali  phosphates  and 
other  salts.  A  few  simple  experiments  may  be  made  to  show 
some  of  the  properties  of  the  common  commercial  extracts. 

Ex.  Heat  a  little  of  the  solid  extract  on  a  piece  of  porcelain  until  it 
is  reduced  to  a  char.  Extract  this  with  dilute  nitric  acid,  filter  and  divide 
the  filtrate  into  two  portions.  In  one  test  for  phosphates  by  the  addition 
of  ammonium  molybdate  and  in  the  other  for  potassium  salts  by  the 
flame  test.     Both  reactions  should  be  very  distinct. 

Ex.  Dissolve  20  grams  of  extract  in  water  to  make  about  200  cubic 
centimeters.  A  nearly  clear  solution  should  be  obtained,  showing  absence 
of   fat  or  coagulated  protein.     To   a   few  cubic   centimeters   add   enough 


346  PHYSIOLOGICAL    CHEMISTRY. 

weak  acetic  acid  to  give  a  slight  reaction,  and  boil.  If  a  precipitate  forms, 
which  is  rarely  the  case,  albumin  is  shown. 

With  50  cc.  of  the  liquid  make  the  albumose  test.  Add  to  it  finely 
powdered  zinc  sulphate  as  long  as  it  dissolves  on  stirring.  On  saturating 
the  solution  completely  a  flocculent  precipitate  gradually  settles.  This  is 
essentially  the  "  albumose "  fraction  and  may  contain  a  little  gelatin. 
After  24  hours  filter,  and  test  the  filtrate  for  peptone  by  the  biuret  reac- 
tion ;  this  is  generally  negative. 

Use  the  remainder  of  the  original  solution  for  the  recognition  of  crea- 
tine. Add  to  it  carefully  a  solution  of  basic  acetate  of  lead  as  long  as 
a  precipitate  forms.  This  will  carry  down  phosphates,  sulphates  and 
other  compounds  forming  insoluble  combinations  with  it,  but  not  creatine. 
A  slight  excess  of  the  lead  must  be  added  to  insure  complete  precipitation. 
This  can  be  determined  by  allowing  the  first  formed  precipitate  to  settle 
and  adding  more  reagent  as  necessary.  Finally  filter,  and  remove  the 
excess  of  lead  by  passing  in  hydrogen  sulphide.  Filter  again,  and  remove 
as  much  as  possible  of  the  excess  of  sulphide  used,  by  shaking.  Then 
concentrate  the  liquid  to  a  small  volume  by  slow  evaporation  on  the  water- 
bath  and  allow  it  to  stand  a  day  or  more  in  a  cool  place  for  crystalliza- 
tion of  the  creatine.  Pour  off  the  supernatant  liquid  and  wash  the  fine 
crystals  obtained  with  a  little  strong  alcohol  in  which  creatine  is  but 
slightly  soluble. 

Ex.  Dissolve  the  creatine  in  a  little  hydrochloric  acid  and  evaporate 
the  solution  slowly  to  dryness  on  the  water-bath.  This  action  converts 
creatine  into  creatinine.  Dissolve  the  residue  in  a  little  water  and  divide 
the  solution  into  two  parts.  To  one  add  a  solution  of  zinc  chloride, 
which  produces  a  white  crystalline  precipitate  containing  the  creatinine- 
zinc  chloride,  (GH7N30)2ZnCl2.  The  character  of  the  crystals  can  be 
seen  under  the  microscope.  To  the  other  part  of  the  solution  add  a  few 
drops  of  a  dilute  solution  of  sodium  nitroprusside  and  then,  drop  by  drop, 
dilute  solution  of  sodium  hydroxide.  This  gives  a  ruby  red  color  which 
fades  to  yellow.  Add  enough  acetic  acid  to  change  the  reaction  and 
warm.  The  color  becomes  green  and  finally  blue.  This  is  known  as 
Weyl's  reaction.     The  blue  color  finally  obtained  is  Prussian  blue. 

Ex.  The  mother  liquor  left  after  crystallizing  the  creatine  contains 
traces  of  xanthine  bases.  Add  enough  ammonia  to  give  an  alkaline  reac- 
tion and  filter.  Then  add  a  few  drops  of  ammoniacal  solution  of  silver 
nitrate  which  precipitates  the  several  substances  in  flocculent  form. 

BONE  AND  GELATIN. 

In  the  moist  bone  as  it  exists  in  the  body  the  water  and 
solids  are,  in  the  mean,  in  about  the  proportion  of  one  to  two. 
In  very  young  persons,  however,  the  water  is  in  greater  ex- 
cess, while  with  age  the  solids  increase.  The  solid  matter 
consists  roughly  of  i  part  of  organic  matter  to  2  of  mineral. 


CHEMISTRY    OF    THE    BONES.  347 

THE  ORGANIC  MATTER  OR  OSSEIN. 

The  crude  organic  substance  in  the  bone  is  commonly 
called  ossein;  it  may  be  extracted  with  hot  water  and  forms 
a  gelatinous  mass  on  cooling.  But  fuller  investigations  show 
that  this  ossein  is  not  a  single  substance,  as  several  different 
constituents  may  be  separated  by  proper  solvents.  These  are, 
however,  closely  related  substances  and  for  our  present  pur- 
pose they  may  all  be  considered  as  practically  identical  with 
the  collagen  or  glue-forming  substance  of  the  connective  tis- 
sues. The  conversion  of  the  ossein  or  collagen  into  gelatin 
appears  to  be  a  hydration  process,  as  at  a  higher  temperature 
the  reverse  operation  takes  place.  The  preparation  and  prop- 
erties of  bone  gelatin  may  be  illustrated  experimentally : 

Ex.  Clean  a  long,  slender  bone  (best,  a  rib),  and  immerse  it  in  dilute 
hydrochloric  acid  of  about  ten  per  cent  strength.  Let  it  remain  several 
days.  At  the  end  of  this  time  remove  the  bone  from  the  acid  and  observe 
that  it  has  lost  its  rigidity  and  has  become  very  flexible.  It  may  be  even 
possible  to  tie  it  in  a  knot.  Wash  the  elastic  mass  several  times  in  fresh 
water  to  remove  all  the  hydrochloric  acid,  then  with  a  little  dilute  sodium 
carbonate  solution  followed  by  more  water,  and  finally  boil  it  with  a  small 
amount  of  pure  water.  By  heating  it  long  enough  the  ossein  becomes 
converted  into  gelatin,  which  solidifies,  on  cooling,  to  a  jelly. 

By  boiling  the  bone  ossein  under  pressure  the  formation  of  the  gelatin 
is  very  much  hastened. 

The  solution  as  obtained  above  may  be  used  for  tests  such  as  were 
•described  in  Chapter  V,  under  Gelatin. 

THE  MINERAL  MATTER  IN  BONES. 

We  are  not  able  to  say  exactly  how  the  mineral  elements 
are  combined  in  the  moist  fresh  bone.  Our  knowledge  of 
these  combinations  is  practically  limited  to  what  we  can  learn 
by  a  study  of  the  residue  left  on  burning  the  bone  completely, 
known  as  boneash.  This  is  a  white  powder  containing  the 
non-volatile  compounds,  of  which  calcium  phosphate  is  the 
most  important.  The  following  table  shows  the  average  com- 
position of  human  boneash : 


348  PHYSIOLOGICAL    CHEMISTRY. 

Calcium    phosphate    857  per  cent. 

Magnesium   phosphate    15 

Calcium  carbonate    no 

Calcium  fluoride  and  chloride 1.0 

Ferric   oxide    0.8 

1 00.0 

The  presence  of  calcium,  magnesium  and  phosphoric  acid 
may  be  shown  in  the  weak  hych-ocliloric  acid  extract  of  the 
bone  described  above. 

Ex.  To  a  few  cubic  centimeters  of  the  filtered  solution  add  some 
ammonium  molybdate  solution.  In  a  short  time  a  yellow  precipitate 
appears,  indicating  presence  of  a  phosphate,  as  familiar  to  the  student 
from  the  reactions  of  qualitative  analysis. 

Ex.  To  a  few  cubic  centimeters  of  the  solution  add  solution  of  sodium 
acetate  until  a  distinct  odor  of  acetic  acid  persists.  Then  add  some  solu- 
tion of  ammonium  oxalate,  which  produces  a  white  precipitate  of  calcium 
oxalate. 

Ex.  To  another  portion  of  the  hydrochloric  acid  solution  add  ammonia 
until  a  good  alkaline  reaction  is  obtained.  A  white  precipitate  of  calcium 
and  magnesium  phosphates  settles  out.  Filter,  and  to  the  filtrate  add 
some  ammonium  oxalate  solution.  A  further  precipitate  appears.  This 
is  calcium  oxalate  and  proves  that  the  original  solution  contains  calcium 
in  excess  of  that  combined  as  phosphate.  The  calcium  of  the  carbonate, 
fluoride  and  chloride  appears  here. 

Ex.  To  detect  the  small  amount  of  magnesium  requires  greater  care. 
To  another  and  relatively  large  portion  of  the  acid  solution  add  enough 
ammonia  to  give  an  alkaline  reaction,  and  then  acidify  slightly  with  acetic 
acid.  This  dissolves  everything  except  ferric  phosphate,  which  may  be 
filtered  off  and  tested  for  iron.  To  the  filtrate  add  enough  ammonium 
oxalate  to  precipitate  all  the  calcium  as  oxalate.  Separate  this  after  long 
standing  by  means  of  close-grained  filter  paper.  In  the  clear  filtrate  the 
magnesium  may  be  thrown  down  with  the  phosphoric  acid  still  present, 
by  the  addition  of  ammonia  water  in  slight  excess. 

Bone  Marrow.  The  pure  marrow^  consists  largely  of  fat 
in  which  olein  is  abundant;  cholesterol  is  present  and  some 
nitrogenous  extractive  substances,  which,  however,  have  not 
been  very  thoroughly  examined. 

CARTILAGE. 

Collagen  is  probably  the  most  abundant  substance  in  the 
cartilaginous  tissue  where  it  exists  mixed  or  combined  with 
several   other  bodies,   of   which   these  have  been   described : 


CARTILAGE    AND    KERATIN    BODIES. 


349 


chondromncoid,  chondroitin-siilphuric  acid  and  an  albiiininoid. 
The  nature  of  crude  collagen  has  been  explained,  and  in 
Chapter  V  the  somewhat  obscure  chemistry  of  the  chondroitin- 
sulphuric  acid  has  been  outlined.  Of  the  nature  of  the 
chondromucoid  little  is  known  definitely;  it  has  been  held  by 
some  writers  to  be  merely  a  combination  of  part  of  the  colla- 
gen with  the  salts  of  the  complex  ethereal  sulphuric  acid  men- 
tioned, while  Morner,  who  first  described  it,  held  it  for  a  dis- 
tinct body  somewhat  allied  tO'  mucin.  His  analyses  showed 
C  47.30,  H  6.42,  N  12.58,  S  2.42,  O  31.28.  The  sulphur  is 
probably  all  in  the  ethereal  combination  and  on  incineration 
of  the  cartilage  the  ash  is  found  to  contain  a  very  large  amount 
of  alkali  sulphate. 

Chondromucoid  as  separated  is  insoluble  in  water  alone,  but 
with  a  little  alkali  forms  a  thick  solution,  which  is  precipitated 
by  acids.  Stronger  acids  bring  about  a  cleavage  with  separa- 
tion of  the  chondroitin  sulphuric  acid.  The  weak  alkali  solu- 
tions are  precipitated  by  metallic  salts,  but  most  of  the  other 
protein  reactions  fail.  The  ethereal  sulphate  group  seems  to 
prevent  the  ordinary  precipitations. 

The  albuminoid  substance  is  not  well  characterized  but  is 
insoluble  in  water,  and  in  weak  acids  or  alkalies.  It  undergoes 
gastric  digestion.  This  protein  is  said  to  be  found  in  old 
cartilage  only,  and  is  absent  in  young  cartilage. 

KERATIN  BODIES. 

Compounds  of  the  keratin  group  occur  in  hair,  the  finger 
nails  and  horn.  They  resemble  the  proteins  but  contain 
rather  large  amounts  of  sulphur,  as  shown  by  these  analyses, 
which  are  of  keratin  from  several  sources : 


Hair. 

Nails. 

Horn. 

c 

50.65 

6.36 

17.14 

20.85 

5.00 

51.00 

6.94 

17-51 

21.75 

2.80 

51.03 
6.80 

H 

N  

16.24 
22.51 

3-42 

0 

S 

350  PHYSIOLOGICAL    CHEMISTRY. 

The  sulphur  in  hair  rs  in  part  loosely  combined  and  may 
be  split  off  easily  by  reagents,  alkalies  for  example.  The  ash 
of  hair  is  rich  in  sulphates  and  contains  also  silica  and  other 
mineral  substances.  Much  of  the  ash  may  l^e  removed  by 
washing  the  hair  with  weak  acids,  following  treatment  with 
ether  and  alcohol  to  remove  fatty  and  other  soluble  substances. 
The  purified  "  keratin  "  thus  secured  gives  results  like  the 
above  on  analysis. 

Horn  and  nails  contain  along  with  the  insoluble  keratin 
insoluble  salts,  mainly  phosphate  of  calcium,  which  stiffen 
them.  From  very  fine  horn  shavings  these  salts  may  be  dis- 
solved out  by  acids,  leaving  a  soft  flexible  keratin. 


SECTION    IV. 

THE   END  PRODUCTS   OF   METABOLISM. 
EXCRETIONS.     ENERGY   BALANCE. 

CHAPTER    XIX. 

THE  NITROGENOUS  EXCRETION.     URINE. 

Having  considered  in  the  foregoing  pages  the  substances 
used  in  the  nutrition  of  the  body,  the  agencies  of  nutrition, 
and  the  general  character  of  the  products  formed,  we  come 
now  to  a  short  study  of  the  waste  products  rejected  by  the 
body  after  it  has  assimilated  and  used  the  nutritives  furnished 
to  it.  The  food-stuffs  which  the  animal  can  utilize  are  com- 
paratively complex,  but  consist  essentially  of  the  members  of 
the  three  groups,  the  fats,  carbohydrates  and  proteins.  The 
theoretically  simplest  waste  or  oxidation  products  of  these  are 
nitrogen,  carbon  dioxide  and  water,  but  in  the  animal  organ- 
ism the  breaking  down  does  not  go  so  far.  While  from  fats 
and  carbohydrates  essentially  only  water  and  carbon  dioxide 
are  formed,  the  protein  metabolism  is  not  carried  to  the  elim- 
ination of  nitrogen,  but  ends  with  the  formation,  largely,  of 
urea,  a  body  in  a  way  related  to  the  theoretical  end  products, 
but  which  would  call  for  three  more  atoms  of  oxygen  to  com- 
plete oxidation. 

The  nitrogen  metabolism  involves  some  extremely  interest- 
ing problems  which  are  still  far  from  complete  solution. 
From  the  older  point  of  view  urea  was  considered  the  one  nor- 
mal end  point  in  the  chain  of  katabolic  reactions,  and  the  other 
nitrogenous  bodies  found  in  the  urine,  such  as  uric  acid  and 
creatinine,  were  looked  upon  as  substances  which  in  some  way 
had  accidentally  escaped  the  fate  due  them.  This  view  is 
doubtless  incorrect,  as  we  have  good  reason  to  believe  that 

351 


352  PHYSIOLOGICAL    CHEMISTRY. 

uric  acid  is  not  a  step  in  the  ordinary  protein  metabolism,  but 
is  a  derivative  of  certain  substances  only,  which  break  down 
to  a  limited  degree.  The  amount  of  uric  acid  which  could 
be  formed  in  this  way  would  not  be  very  large  at  most.  In 
the  metabolism  of  nitrogen,  therefore,  a  number  of  normal 
end  products  must  be  considered  and  these  will  be  discussed 
in  the  next  few  pages.  It  \\'\\\  be  well  to  begin  with  the  con- 
sideration of  the  urine  as  a  whole,  as  all  these  substances  are 
eliminated  through  that  channel. 

THE  GENERAL  COMPOSITION  OF  URINE. 

The  work  of  the  kidneys  in  the  discharge  of  the  urine,  or 
more  properly  the  separation  of  its  constituents  from  the  blood, 
is  usually  spoken  of  as  one  of  excretion.  But  something  more 
than  simple  elimination  of  worthless  products  is  here  con- 
cerned :  the  work  done  by  these  organs  is  in  part  secretory, 
as  certain  synthetic  reactions  are  beyond  question  carried  out 
here.  Years  ago  Bunge  and  Schmiedeberg  demonstrated  the 
synthesis  of  hippuric  acid  from  benzoic  acid  and  glycocoll  in 
the  kidney,  and  since  then  other  changes  have  been  brought  to 
light.  Further  than  this,  the  peculiar  mechanism  of  the  kid- 
ney accomplishes  another  very  remarkable  thing.  The  blood 
circulating  through  the  kidney  contains  valuable  material  to 
be  saved  as  well  as  worthless  substances  to  be  rejected. 
Toward  all  these  constituents  the  epithelial  cells  of  the  kidney 
tubules  exercise  a  sort  of  selective  treatment.  The  proteins, 
which  are  colloids,  are  retained  by  the  blood,  but  the  sugar, 
which  is  a  crystalloid,  and  very  soluble,  is  retained  also  unless 
its  concentration  passes  a  certain  limit.  The  soluble  salts  are 
in  part  passed  through  the  kidneys  and  in  part  retained  by  the 
blood,  with  the  final  result  of  maintaining  a  very  nearly  con- 
stant osmotic  pressure  in  that  fluid.  How  this  is  done  we 
cannot  say.  It  is  indeed  a  problem  of  physiology  and  his- 
tolog}'  rather  than  of  chemistry.  We  know  only  this,  that  the 
selective  absorption  and  control  of  the  blood  concentration  are 


THE   NITROGENOUS    EXCRETION.       URINE.  353 

perfectly  automatic.  When  the  osmotic  pressure  of  certain 
constituents  is  increased  beyond  a  pretty  definite  hmit,  the  fil- 
tering mechanism  in  the  kidney  for  those  constituents  becomes 
active  and  the  excess  is  alloAved  to  pass.  The  simple  laws  of 
diffusion  and  osmotic  pressure  do  not  help  us  greatly  in  ex- 
plaining the  actions  of  the  kidneys  where  the  flow  of  excreted 
substances  is  usually  from  a  level  of  low  concentration  to  one 
of  higher.  Attempts  have  been  made  to  compare  the  separat- 
ing medium  between  the  urine  and  the  blood  to  a  semiperme- 
able membrane,  but  the  comparison  is  very  imperfect  unless 
the  degree  of  impermeability  be  specially  limited  for  each  sub- 
stance passing  from  the  blood  to  the  urine.  The  limitation 
would  have  to  account  for  a  concentration  of  salt  from  about 
0.6  per  cent  in  the  blood  to  over  i.o  per  cent  in  the  urine, 
while  for  urea  the  concentration  would  change  from  about 
0.05  per  cent  or  lower  to  over  2.0  per  cent,  that  is  forty  fold. 
Limitations  as  wide  as  these  render  the  comparison  of  little 
practical  service. 

Percentage  Variations.  It  is  not  possible  to  speak  of  the 
mean  strength  of  normal  urine  since  the  variations  are  ex- 
tremely irregular,  depending  in  health  on  a  great  many 
factors.  The  volume  excreted  daily,  as  stated  in  the  books,  is 
usually  given  much  too  high  for  the  conditions  obtaining  in 
the  United  States.  In  place  of  the  1,500  cc.  as  found  in  most 
of  the  foreign  works  we  should  take  1,150  to  1,200  cc.  as 
nearer  the  average  excretion  for  24  hours.  In  some  hundreds 
of  examinations  made  by  the  writer  in  the  last  few  years  on 
people  of  both  sexes  engaged  in  various  occupations  the  aver- 
age volume  comes  within  these  limits. 

A  number  of  complete  analyses  of  urine  are  found  in  the 
literature,  but  in  most  of  them  the  uric  acid  content  is  placed 
too  low  because  of  the  faulty  methods  of  determination  for- 
merly employed.  In  the  following  table  are  given  some  results 
obtained  in  the  author's  laboratory  in  which  the  recognized 
sources  of  error  have  been  avoided  as  far  as  possible.  In  each 
case  the  mixed  urine  of  the  24  hours  was  used,  and  the  am- 
24 


354 


PHYSIOLOGICAL    CHEMISTRY. 


monia  and  urea  determinations  were  made  without  delay,  so 
as  to  avoid  error  through  decomposition  of  the  urine.  The 
results  are  given  in  terms  of  the  radicles  as  found  by  the 

analyses : 

Table  of  Complete  Urine  Analyses. 

Nos.  I,  2  and  3  mixed  diet.     Nos.  4,  5  and  6  vegetarian.     Results  in 
grams  per  100  cc. 


20° 


Specific  gravity,      - 1.024 

4 

Potassium,  K 

Sodium,  Na |   o 

Calcium,  Ca o 


1.020        1.026 


Magnesium,  Mg 

Ammonium,  NH^ 

Chlorine,  CI 

Phosphoric  acid,  FO^. 
Sulphuric  acid,  SO^.... 

Urea,  CON,H, 

Uric  acid  (C^H^NPs) 
Creatinine,  C^H^NjO.. 


1980 

3820 
0104 
0092 
0900 
7366 

H43 

1640 

85 
0641 

1750 


0.2455 

0.3710 

0.0086 

0.0121 

0.0840 

0.7160 

0.1742 

0.1964 

2.60 

0.0691 

0.1790 


o.  2604 

0.4647 

0.0132 

0.0152 

0.1080 

0.7739 

0.3135 

0.3230 

2.97 

0.0876 

o.  1414 


1. 017 
O.I4I6 

0-3419 

o.  003 1 
0.0144 
0.0800 

0.5254 

0.2062 
0.1965 

2.  I  I 

0.0549 

0.0252 


1.028 

0.4872 

0.3578 
0.0164 
0.0225 
0.0745 
0.8165 
O.3III 
0.2790 

2.95 

0.0769 

0.0910 


1. 021 

0.3869 
0.2705 
0.0130 
0.0341 
0.0576 
0.5893 
0.2230 

o.  2039 

2.80 
0.0838 

o.  1050 


The  urines  numbered  i,  2  and  3  were  from  well-nourished 
male  students;  numbers  4,  5  and  6  from  men  who  had  been 
for  a  long  period  consistent  vegetarians,  consuming  only 
bread,  fruits,  vegetables  and  nuts  and  a  very  small  amount  of 
milk.  The  average  volume  for  i,  2  and  3  was  nearly  1.200 
cc,  for  4,  5  and  6  about  1,000  cc.  No  characteristic  differ- 
ences are  readily  apparent.  In  the  above  table  no  determina- 
tions of  the  traces  of  xanthine  bodies  are  included  and  there 
are  also  no  carbonic  acid  determinations.  For  comparison  be- 
tween the  bases  and  acids  it  is  convenient  to  calculate  the  first 
in  terms  of  Na  and  the  latter  in  terms  of  CI,  assuming  that 
the  acids  all  act  to  form  theoretically  neutral  salts.  The  re- 
sults of  such  a  calculation  are  sfiven  in  the  table  below. 


Number. 

Metals  in  Terms  of       Acids  in  Terms 
Na.                1              ofCl. 

Chlorine  Theoretic- 
ally Necessary 
for  Na. 

Apparent  Excess 
of  Acids  as  CI. 

I 

2 

3 
4 
5 
6 

0.6430                         I. 0134 
0.6560                        1.0860 
0.8002                         I. 4017 
0.5586                        0.9274 
0.8015                         1.4044 
0.6519                         1.0259 

0.9924 
1.0126 
1.2350 
0.8622 
1.2371 
1.0062 

0.0210 
0  0734 
0.1667 
0.0652 
0.1673 
0.0197 

THE    NITROGENOUS    EXCRETION. 


URINE. 


JDD 


The  acid  radicles  appear  to  be  greatly  in  excess  but  this 
comes  mainly  from  the  method  of  calculation.  The  uric  acid 
and  phosphoric  acid  in  the  urine  are  in  large  part  undoubtedly 
combined  as  acid  salts.  By  calculating  the  phosphoric  acid  as 
salts  of  the  type  M2HPO4.  and  the  uric  acid  as  forming  salts 
of  the  type  MHCgHoN^Oo,  this  acid  excess  disappears  in  all 
cases  except  No.  5  and  a  slight  basic  excess  results.  Unques- 
tionably the  carbonic  acid  present  should  be  considered  in 
order  to  secure  a  complete  picture  of  the  combinations.  It 
must  also  be  remembered  that  a  small  part  of  the  sulphuric 
acid  always  exists  in  the  form  of  so-called  ethereal  sulphates 
which  are  not  considered  in  the  above  figures. 

The  calculation  of  the  form  of  combination  of  the  bases 
and  acids  from  the  results  of  analyses  is  always  arbitrary  but 
is  sometimes  of  value  in  suggesting  the  possible  salt  com- 
pounds. Such  a  computation  made  from  the  analytical  results 
above  gives  the  following  table,  in  which  for  comparison  neu- 
tral salts  are  considered  as  formed.  This  table  embraces  the 
inorganic  substances  only.  If  the  uric  acid  were  included  the 
acid  excess  would  appear  as  in  the  last  table  given.  The 
values  are  in  grams  per  100  cc.  as  before: 


I 

2 

3 

+ 

5 

6 

NaCl 

KCl 

0.9716 
0.3089 

0.9436 
0.3013 

1.1819 
0.1191 

0.8696 

0.9100 

0-5554 

O.6S80 
0.3610 

(NH,,,SO,, 

KjPO^ 

(NHJ3PO,. 
Ca3(P0J,.  . 
Mg3(P0J,. 
Mg,  excess.. 
PO,,  excess . 


0.0814 
0.1646 

0.1244 
0.0269 
0.0255 
0.0022 


0.195 1 
0.1222 

0.1399 
0.0222 
0.0440 


0.4411 
0.1098 

0.2155 
0.0341 
0.0553 


0.3154 
0.0312 

0.1973 
o.  0080 
0.0524 


0.4363 
0.0527 

0-1653 
0.0423 
0.0818 


0.0395  ,  0.1 152   0.0375  I  0.1204 


0.3704 

0.0565 
0.1591 
0.0336 
0.1062 
0.0055 


The  figures  show  pretty  well  the  relative  importance  of 
sodium,  potassium  and  ammonium  salts.  In  all  cases  there  is 
sufficient  chlorine  to  combine  with  all  the  sodium,  and,  in  all 
except  the  fourth  urine,  with  a  good  part  of  the  potassium  also. 
Two  of  the  vegetarian  urines  are  very  high  in  potassium  salts, 
probably   from  peculiarities   in   the   diet   consumed.     Certain 


356 


PHYSIOLOGICAL    CHEMISTRY, 


vegetables  and  fruits  contain  relatively  large  quantities  of 
potassium  salts  of  organic  acids,  and  such  substances  may 
have  been  consumed  here.  It  is  important  to  note  further  the 
amounts  of  ammonia  in  these  urines.  This  form  of  excretion 
has  been  frequently  overlooked  in  calculating  urine  analyses, 
but  as  will  appear  below  the  nitrogen  in  it  makes  up  an  appre- 
ciable fraction  of  the  total  excretion.  In  this  place  it  will  be 
well  also  to  call  attention  to  the  ratio  of  urea  to  uric  acid, 
which  was  formerly  frequently  stated  too  high  because  of 
faulty  methods  used  in  determining  the  latter,  as  referred  to 
above.     From  the  abo\'e  tables  it  appears  in  this  way : 


I 

« 

3 

4 

5 

6 

Urea 
Uric  Acid 

43.8 

37-2 

33-6 

38.3 

37-9 

32.9 

We  may  turn  now  to  a  consideration  of  individual  constit- 
uents in  the  excretion. 


THE  EXCRETION  OF  ALKALI  SALTS. 

The  alkali  salts  found  in  the  urine  come  from  the  sodium 
chloride  consumed  as  such  in  salted  food,  and  in  part  from 
potassium  salts  in  the  juices  of  meat  and  in  vegetables.  In  the 
analysis  of  the  ash  of  muscle  given  some  pages  back  chlorine 
as  well  as  potassium  is  shown.  Chlorine  is  found,  although 
usually  in  small  amount,  in  the  ash  of  all  vegetable  substances. 
In  the  latter,  however,  especially  in  the  cereals,  potassium 
phosphate  is  the  characteristic  constituent  of  the  ash.  On  a 
cereal  diet  we  should  expect  the  urine,  in  consequence,  to  show 
a  relatively  high  potash  and  phosphoric  acid  content.  The 
ash  of  potatoes  contains  in  the  mean  over  60  per  cent  of  potas- 
sium oxide  while  the  chlorine  is  in  excess  of  the  sodium. 
With  a  mixed  diet  therefore  the  composition  of  the  alkali  salts 
in  the  urine  must  be  variable  and  difficult  of  explanation.  As 
the  alkali  compounds  are  practically  all  soluble  they  are  ex- 
creted almost  solely  by  the  urine  and  to  a  small  extent  only  by 


THE    NITROGENOUS    EXCRETION.       URINE.  3  57 

the  feces.  The  analysis  of  the  urine  gives  us  then  in  ordinary- 
cases  a  fairly  accurate  measure  of  the  alkali  metals  taken  in 
with  our  food  and  drink;  in  normal  condition  there  is  no  ac- 
cumulation of  alkali  salts  in  the  body. 

CALCIUM  AND   MAGNESIUM   COMPOUNDS. 

The  full  significance  of  these  salts  in  the  urine  we  can  not 
explain,  since  without  complete  analyses  of  the  feces  we  do 
not  know  the  relation  of  the  excreted  to  the  ingested  alkali- 
earths.  Our  natural  drinking  waters  contain  usually  quite 
appreciable  amounts  of  these  salts,  with  those  of  calcium  in 
excess  as  a  rule.  In  Lake  Michigan  water,  for  example,  we 
have  about  125  milligrams  per  liter  of  these  salts  as  carbonates, 
but  in  our  common  animal  and  vegetable  foods  we  consume 
daily  much  greater  quantities  than  we  could  get  from  water. 
The  ash  of  wheat  contains  about  12  per  cent  of  magnesia  and 
3  per  cent  of  lime,  while  in  the  ash  of  muscle  we  have  over  3 
per  cent  of  magnesia  and  between  2  and  3  per  cent  of  lime. 
500  grams  of  lean  meat  would  furnish  us  then  with  over  150 
milligrams  of  magnesia  and  with  something  less  than  that 
amount  of  lime. 

But  only  fractions  of  these  compounds  find  their  way  into 
the  urine.  In  the  original  foods  they  exist,  in  part  at  least, 
in  insoluble  forms.  AVhile  some  of  these  substances  may  be 
dissolved  in  the  stomach  the  conditions  are  reversed  in  the 
intestines,  and  insoluble  phosphates,  carbonates  and  sulphates 
are  lost  with  the  feces.  There  has  been  much  discussion  as  to 
the  exact  naturCjOf  the  calcium  and  magnesium  salts  excreted. 
In  a  measure  the  discussion  is  fruitless,  as  we  must  certainly 
admit  the  free  exchange  of  ions  in  solution.  Under  ordinary 
conditions  the  acid  ions  of  the  urine  appear  to  be  in  slight 
excess  of  the  metals,  which  prevents  precipitation  of  insoluble 
phosphates,  for  example.  Temperature  plays  a  very  impor- 
tant part  in  the  problem  of  the  stability  of  the  calcium  and 
magnesium  compounds  in  the  urine,  and  the  problem  is  further 
complicated  by  the  presence  of  uric  acid,  the  peculiar  behavior 
of  which  will  be  touched  upon  below. 


358  PHYSIOLOGICAL    CHEMISTRY. 

THE   SULPHUR   EXCRETION. 

The  sulphur  in  the  urine  is  found  in  several  compounds  and 
comes  from  different  sources.  A  small  part  has  its  origin  in 
traces  of  sulphates  taken  directly  in  food  and  natural  waters; 
some  comes  from  the  sulphur  existing  in  peculiar  combina- 
tions in  certain  vegetables,  while  the  largest  part  has  its  origin 
in  the  sulphur  of  proteins,  which  undergoes  more  or  less  com- 
plete oxidation  before  elimination  through  the  kidneys.  Most 
of  this  sulphuric  acid  of  oxidation  combines  with  alkalies  for 
elimination;  if  fixed  alkalies  are  deficient  ammonium  sulphate 
is  formed  and  this  ammonia  therefore  escapes  the  natural  oxi- 
dation to  urea. 

It  has  been  explained  in  an  earlier  chapter  that  in  the  pu- 
trefactive changes  taking  place  in  the  intestines  certain  phenol 
bodies  are  split  off  from  proteins  there  remaining,  or  perhaps 
in  most  cases  from  the  unabsorbed  protein  derivatives.  A 
very  considerable  part  of  these  phenol  bodies  escapes  with  the 
feces,  but  another  portion,  often  much  increased  in  disease,  is 
absorbed  by  the  blood  vessels  from  the  lower  intestine  and  car- 
ried to  the  liver  where  combination  with  sulphuric  acid  is 
effected,  probably  through  some  kind  of  enzymic  action. 
The  ethereal  sulphate  so  formed  is  discharged  finally  with  the 
urine.  The  fraction  of  the  sulphuric  acid  so  voided  is  ex- 
tremely variable,  reaching  sometimes  20  per  cent  of  the  whole. 
The  most  abundant  of  these  combinations  are  salts  of  phenyl-, 
cresyl-,  indoxyl-,  and  skatoxyl-sulphuric  acid,  the  structural 
relations  of  which  are  shown  by  the  following  formulas : 


CsH^O^  CH3.C«H,0) 


CH 
HC       C — C.O.SO,OH 


HC       C      CH 

C      N 
H      H 

Phenyl-sulphuric  acid       Cresyl-sulphuric  acid  Indoxyl-sulphuric  acid 

Skatoxyl-sulphuric  acid  is  the  methyl  derivative  of  indoxyl- 
sulphuric  acid.      Phenyl-  and  cresyl-sulphuric  acids  are  fre- 


THE    NITROGENOUS    EXCRETION.       URINE.  359 

quently  called  phenol-  and  cresol-sulphuric  acids,  but  the 
former  names  are  preferable.  The  alkali  salts  of  indoxyl- 
sulphuric  acid  are  known  as  indican,  the  appearance  of  which 
in  the  urine  in  quantities  above  minute  traces  is  an  indication 
of  the  existence  of  excessive  putrefactive  reactions  in  the  intes- 
tines. The  observation  is  therefore  of  value  in  diagnosis. 
Accurate  methods  are  known  for  the  recognition  of  the  sub- 
stance in  the  urine. 

In  addition  to  the  oxidized  or  sulphate  sulphur  in  the 
urine  there  are  always  traces  of  so-called  "neutral"  sulphur 
compounds  present.  According  to  various  authorities  the  sul- 
phur in  these  compounds,  which  are  all  complex  organic 
bodies,  may  make  up  12  to  25  per  cent  of  the  total  sulphur. 
The  sulphur  bodies  here  in  question  are  not  easily  recognized 
in  most  cases  and  the  existence  of  several  of  them  described  in 
the  journals  is  yet  to  be  demonstrated.  The  fact  of  the  excess 
of  sulphur  over  that  contained  in  the  mineral  and  ethereal 
sulphates  may  be  easily  shown  by  determining  the  total  sul- 
phate sulphur  directly,  and  then  after  complete  oxidation  of 
the  urine  residue  obtained  by  evaporation.  Among  the  bodies 
containing  the  unoxidized  sulphur  these  have  been  recognized 
with  greatest  certainty:  cystin,  taurin,  oxyproteic  acid  and 
alloxyproteic  acid,  of  which  something  will  be  said  below.  It 
is  not  possible  to  give  the  exact  significance  of  these  bodies  in 
the  urine,  but  they  doubtless  represent  imperfect  or  incomplete 
oxidation  stages  of  the  original  proteins. 

THE  PHOSPHORUS   EXCRETION. 

As  mentioned  above  some  of  our  foods  are  very  rich  in 
phosphates  or  phosphate-furnishing  material.  This  is  espe- 
cially true  of  the  cereals.  The  phosphates  formed  by  oxida- 
tion of  these  substances  are  mainly  eliminated  with  the  urine, 
and  in  traces  with  the  feces.  A  very  considerable  portion  of 
the  urinary  phosphoric  acid  comes  from  this  source.  An- 
other portion  comes  from  the  breaking  down  of  the  nucleinic 
acid  of  the  cell  substance  and  from  the  so-called  phospho- 


T,6o 


PHYSIOLOGICAL    CHEMISTRY. 


globulins  or  nucleoalbumins.  How  the  phosphorus  is  held  in 
these  last-named  bodies  is  not  known,  but  in  the  nucleinic 
acids  it  appears  to  be  in  oxidized  form,  according  to  some 
authorities  as  metaphosphoric  acid,  or  possibly  as  the  pyro- 
acid.  The  excreted  product  is  orthophosphoric  acid,  com- 
bined to  form  salts  of  the  type  ]\IH.P04  or  MoHPO,.  The 
alkali  salts  are  soluble  readily,  while  those  of  calcium  and 
magnesium  are  only  in  part  soluble  in  water.  The  conditions 
of  solubility  in  urine  are  complicated  by  the  presence  of  other 
salts. 

The  amount  of  phosphoric  acid,  as  P.jOs,  excreted  daily 
varies  within  wide  limits:  according  to  the  above  analyses 
between  about  1.3  and  3.5  gm.  If  the  urine  becomes  alka- 
line through  the  fermentation  of  urea  a  very  considerable  part 
of  the  phosphate  may  be  precipitated  in  the  form  of  calcium 
and  magnesium  salts.  One  of  the  commonest  of  these  is  the 
so-called  triple  phosphate,  NH4MgP04  •  6H2O. 

THE  NITROGEN  EXCRETION. 

For  many  reasons  this  excretion  is  the  most  important 
which  we  have  to  consider  in  connection  with  the  urine,  as  it 
gives  us  an  insight  into  some  of  the  fundamental  problems 
in  metabolism.  The  largest  part  of  it  leaves  the  body  as  urea, 
but  the  proportion  excreted  in  other  compounds  cannot  be 
neglected.  Considering  the  four  substances,  ammonia,  urea, 
uric  acid  and  creatinine  only,  we  have  the  following  percent- 
age distribution  in  the  table  given  above : 


1                   ; 

I                  2 

'         1          ' 

5 

6 

N  in  ammonia....        4.60             4.78 
N  in  urea 89.68           8865 

5-41             5-78 

89.30          91.62 

1.90      1       1.72 

3-39     !       0.88 

3.88 

92.14 

1.72 

2.26 

3-16 
92.11 

1.97 

2.76 

N  in  uric  acid....  1        1.44             1.71 
N  in  creatinine...        4.28      j       4.86 

'   100.00      j   100.00 

100.00        100.00 

100.00 

100.00 

The  urea  nitrogen  averages  about  90  per  cent  of  the  whole. 
The  fraction  is  higher  for  the  vegetarian  urines  than  for  the 


THE    NITROGENOUS    EXCRETION.       URINE.  36 1 

others.  In  addition  to  these  four  nitrogenous  bodies  several 
others  are  known  to  be  present  in  very  small  amount;  their 
consideration  would  not  affect  the  relations  given  appreciably. 
But  in  the  last  few  years  a  new  urine  acid  has  been  described 
by  several  writers,  and  especially  by  Bondzynski  and  Gottlieb, 
under  the  name  of  oxyproteic  acid,  which  appears  of  more 
importance.     This  substance,  to  which  the  formula 

was  given,  but  not  definitely  proven,  is  very  soluble  and  hard 
to  separate  or  recognize.  Hence  the  fact  that  it  so  long  es- 
caped notice.  According  to  the  authors  mentioned  it  is  ex- 
creted to  the  extent  of  3  to  4  grams  daily.  Its  nitrogen  would 
therefore  make  up  2  to  3  per  cent  of  the  total  nitrogen  ex- 
creted,  and  if  included  would  modify  somewhat  the  above 

proportions. 

AMMONIA. 

This  represents  a  portion  of  the  protein  disintegration 
which  for  a  number  of  reasons  has  not  been  converted  into 
urea.  The  ammonia  passing  into  the  urine  takes  that  course 
ordinarily  through  combination  with  mineral  or  other  acids 
which  are  not  destroyed  or  may  not  be  destroyed  by  oxida- 
tion. In  any  pathological  increase  of  such  acids,  if  there  is 
not  enough  fixed  alkali  in  the  blood  to  combine  with  them, 
ammonia  is  split  off  from  protein  derivatives  in  quantity  suf- 
ficient to  complete  the  neutralization.  This  may  be  shown 
also  by  the  injection  of  free  mineral  acids  either  directly  or 
with  the  food;  an  increased  elimination  of  ammonia  results. 
It  should  be  expected  therefore  that  the  proportion  of  am- 
monia in  the  urine  would  be  subject  to  marked  fluctuations, 
which  is  indeed  the  case.  Taken  with  other  determinations 
the  estimation  of  ammonia  may  possess  considerable  diagnos- 
tic value,  as  it  measures  to  some  extent  the  excessive  acid 
excretion. 

Ammonia  must  be  determined  in  fresh  urine  only,  since  in 
old  urine  fermentation  changes  soon  produce  large  quantities 
of  the  substance  from  the  breaking  down  of  urea. 


362  PHYSIOLOGICAL    CHEMISTRY. 

UREA. 

The  relation  of  this  substance  to  ammonium  carbonate  has 
been  referred  to  many  times,  but  especially  in  discussing  the 
enzymic  processes  of  the  liver.  The  nutritive  proteins  contain 
many  amino  groups  which  seem  to  be  split  off  in  the  general 
combustion  processes  going  on  in  the  body;  also  a  great  ex- 
cess of  groups  which  oxidize  more  completely  and  yield  car- 
bon dioxide.  The  large  part  of  this  escapes  by  way  of  the 
lungs,  while  another  part  is  evidently  taken  care  of  in  the 
Hver  through  combination  with  the  amino  groups  to  form 
urea.  It  has  just  been  explained  that  normally  some  of  this 
amino  nitrogen  fails  to  take  this  simple  course  because  of  the 
presence  of  strong  acid  radicles,  which  have  great  tenacity  in 
their  combining  relations.  The  ammonium  salts  so  formed 
are  stable  and  cannot  be  worked  over  into  urea. 

It  appears  also  that  the  nitrogen  of  some  other  groups  in 
addition  fails  to  reach  the  urea  stage.  Creatinine  and  uric 
acid  nitrogen  are  not  included  here,  as  these  substances  seem 
to  have  an  independent  origin  which  will  be  discussed  below. 
But  there  are  obscure  compounds  in  the  urine  in  small  amount 
of  which  we  know  but  little,  and  some  of  these  contain  nitro- 
gen. The  oxyproteic  acid  referred  to  above  is  an  illustration. 
What  the  relation  of  this  is  to  urea  we  cannot  say,  but  an 
idea  of  this  kind  suggests  itself :  the  original  protein  complex 
may  contain  certain  groups  which  do  not  fall  an  easy  prey  to 
the  work  of  the  oxidation  enzymes  in  the  body;  they  do  not 
break  down  to  amino  compounds  and  carbon  dioxide,  but  re- 
main intact  as  very  resistant  residues,  and  hence  when  the 
liver  is  reached  they  are  not  in  condition  to  pass  into  the  urea 
stage.  In  the  katabolic  changes  of  protein  it  is  possible  that 
a  number  of  such  resistant  groups  may  be  produced,  and  it  is 
likely  that  the  amount  of  nitrogen  or  other  element  which  so 
escapes  the  normal  end  reaction  depends  largely  on  the 
strength  of  the  enzymic  functions.  These  must  vary  in  dif- 
ferent individuals,  and  hence  sometimes  more  and  sometimes 
less  of  these  resistant,  or  left  over,  residues  will  find  their  way 
into  the  urine. 


THE    NITROGENOUS    EXCRETION.       URINE.  363 

From  this  point  of  view  urea  represents  that  part  of  the 
original  body  nitrogen,  aside  from  the  creatine  and  nuclein 
derivatives,  which  takes  the  normal  course.  It  represents  no 
store  of  practically  realizable  energy,  while  with  some  of  the 
other  bodies  which  escape  in  the  urine  this  is  not  the  case; 
under  more  favorable  conditions  they  might  be  expected  to 
suffer  further  oxidation  with  liberation  of  more  heat.  Such 
ideal  conditions  are  realized  in  some  individuals  more  than 
in  others. 

Urea  may  be  built  up  outside  of  the  body  by  many  syn- 
thetic processes,  but  is  most  easily  prepared  by  the  conversion 
of  ammonium  cyanate,  NH4OCN,  into  the  isomer.  On  evap- 
oration of  a  solution  of  this  salt  the  transformation  into  urea 
is  complete.  Urea  is  very  soluble  in  water,  from  which  it 
may  be  obtained  easily  in  crystalline  form.  Its  solutions  are 
easily  decomposed  by  many  oxidizing  agents  with  formation 
of  water,  carbon  dioxide  and  free  nitrogen,  on  which  behavior 
several  of  the  processes  for  determining  it  are  based.  This 
change  is  brought  about  by  hypochlorites,  for  example,  in  this 

manner : 

CON2H4  +  3NaOCl  =  sNaCl  +  2H2O  -^  CO2  +  N^. 

On  the  other  hand,  urea  may  take  part  in  synthetic  reactions 
and  may  be  combined  to  form  complex  substances,  in  certain, 
cases,  as  will  be  shown  below. 

URIC  ACID  AND  THE  PURINE  BODIES. 

Few  topics  in  physiological  chemistry  have  attracted  more 
attention  than  the  relations  of  uric  acid  to  other  nitrogenous 
products  excreted  in  the  urine,  and  its  behavior  in  relation  to 
disease.  The  importance  of  the  substance  in  this  point  of 
view  has  undoubtedly  been  very  frequently  over-estimated  and 
even  at  the  present  time  clinicians  are  much  divided  as  to  the 
part  it  plays  in  certain  diseases.  This  much  may  be  said  with 
truth,  however,  that  many  of  the  fine  spun  theories  which 
have  been  advanced  by  medical  men  on  the  iu"ic  acid  cjuestion, 


364  PHYSIOLOGICAL    CHEMISTRY. 

and  wliich  have  held  our  attention  for  a  long-er  or  shorter 
period  have  been  founded  on  very  weak  clicniical  evidence, 
and  this,  it  should  be  mentioned,  is  the  real  factor  in  the  case. 
Under  the  older  view,  as  explained  already,  uric  acid  was 
supposed  to  be  but  a  step  in  the  formation  of  urea,  the  normal 
end  product  in  protein  metabolism,  and  numerous  disorders 
were  attributed  to  the  accumulation  of  uric  acid  in  the  blood 
throug'h  some  failure  in  the  final  oxidation  processes.  But  it 
appears  now  from  the  evidence  available  that  uric  acid  is  not 
a  natural  step  in  the  oxidation  of  the  ordinary  proteins ;  it 
does  result,  however,  from  the  breaking  down  of  the  complex 
nucleo  proteids  which  are  represented  to  a  limited  extent  only 
in  the  body,  as  compared  with  the  muscle  proteins,  for  exam- 
ple. The  glandular  organs  rich  in  cells  furnish  the  chief 
amount  of  the  nuclein  complexes.  In  the  katabolism  of  these, 
true  proteins  and  the  residues  rich  in  phosphorus  known  as 
nucleinic  acids  result;  the  proteins  undergo  the  usual  further 
oxidation  probably,  while  the  nucleinic  acids  break  down  into 
a  variety  of  products  of  which  the  xanthine  bases,  ammonia, 
thymine  and  carbohydrate  groups  are  the  most  important. 
The  xanthine  bodies  in  turn  doubtless  give  rise  to  the  uric 
acid.  As  pointed  out  in  Chapter  V  several  nucleinic  acids 
exist :  their  structural  formulas  are  not  known,  but  empirically 
■  these  formulas  have  been  given  to  acids  from  different  sources. 

CoHc2Ni40;5P4  Salmon  milt 

CmHmNhO;6P4  Salmon  milt 

CsgH4,N„0:v,P4  Yeast  cells 

CtiHciNioOsiP*  Wheat  embryo 

The  cleavage  products  of  these  acids  are  not  constant,  since 
from  different  acids  different  xanthine  bases  have  been  made. 
Those  found  in  the  animal  body  are  the  following:  xanthine, 
hypoxanthinc,  guanine,  adenine,  Jieteroxanthine,  paraxanthine 
and  epignanine.  In  order  to  show  the  relations  of  these  com- 
pounds to  uric  acid,  E.  Fischer  proposed  to  consider  them  all 
as  derivatives  of  a  nucleus  group  which  he  called  purine. 
As  the  chemistry  of  these  bodies  is  complex  it  may  be  well 


THE    NITROGENOUS    EXCRETION.       URINE.  365 

to  illustrate  their  relations  by  the  structural  formulas  worked 
out  or  confirmed  by  Fischer.  Starting  with  the  assumed 
purine  nucleus  we  have  these  formulas,  with  the  nucleus  atoms 
numbered,  as  suggested  by  Fischer : 

1  N C  6  N=CH  HN— CO 

I         I      7  I       I     H  I       i 

2  C  5  C— N.  HC     C— N\  OC     C— NH 

I         I         >C8  II      II        >H  I       II        Vh 

3  N C— N^  N— C— N^  I       II        /> 

4      9  HN— C— N 

Purine  nucleus,  CeN^  Purine,  C5H4N4  Xanthine,  CsH^N^Oj 

HN— CO  N=C— NH2  HN— CO 

OC— C— NH  HC     C— N^  HC     C— NH 

I      |l        \C0  l|      II        ^  II      II        \CH 

I      11        /  11      II        /CH  II      II         >« 

HN— C— NH  N— C— N— H  N— C— N 

Uric  acid,  CbH^N^Oj  Adenine,  C5H5N3  Hypoxanthine,  C5H4N4O 

Employing  the  Fischer  nomenclature  these  bodies  have  the 
following  names : 

Adenine    6-aniinopurine 

Hypoxanthine     6-oxypurine 

Xanthine  2,  6-dioxypurine 

Uric  acid   2,  6,  8-trioxypurine 

Guanine    2-amino-6-oxypurine 

As  their  relations  have  been  shown  by  various  syntheses  and 
other  transformations,  and  as  further,  the  xanthine  and  hypo- 
xanthine, adenine  and  guanine  have  been  directly  derived  from 
the  nucleinic  acids,  the  relation  of  uric  acid  to  the  latter  bodies 
is  not  far  to  seek. 

Not  all  of  the  nucleinic  acid  destroyed  can  be  assumed  to 
come  from  body  cell  structures ;  many  of  our  foods  contain  nu- 
cleins  and  these  must  give  rise  to  the  same  derivatives  on  oxi- 
dation without  passing  through,  becoming  part  of,  the  cells  of 
the  glandular  organs  of  the  body.  Accordingly  we  distinguish 
between  endogenous  and  exogenous  purines  and  uric  acid. 
With  the  food  nucleins  eliminated  as  far  as  possible  it  has  been 
found  that  the  uric  acid  excreted  becomes  nearly  constant  and 
bears  a  more  uniform  relation  to  the  urea.     This  indicates  that 


366  PHYSIOLOGICAL    CHEMISTRY. 

the  destruction  of  cell  substance  in  the  body  leads  as  regnlarly 
to  uric  acid  as  does  that  of  muscle  proteins  to  urea.  The  use 
of  rich  protein  foods  does  not  necessarily  occasion  g-reater 
elimination  of  uric  acid.  It  is  only  when  they  contain  appre- 
ciable amounts  of  the  nucleins  that  this  is  the  case.  In  addi- 
tion to  these  facts  it  has  been  found  experimentally  that  the 
oxidation  of  nucleins  outside  the  body  leads  to  the  production 
of  uric  acid  in  small  amount. 

Uric  acid  may  be  obtained  synthetically  by  combining  urea 
with  glycocoll,  and  at  a  high  temperature  it  may  be  decom- 
posed with  production  of  urea,  ammonia,  prussic  acid  and 
other  bodies,  under  different  conditions.  But  little  importance 
is  attached  to  these  facts  at  the  present  time,  but  formerly  they 
were  supposed  to  support  the  view  that  uric  acid  is  a  stage  in 
the  urea  formation  through  which  all  the  katabolic  nitrogen 
should  pass.  Of  greater  interest  is  this  fact  that  when  uric 
acid  is  introduced  into  the  circulation  of  certain  animals  some 
of  it  appears  to  be  destroyed,  and  with  the  production  of  a  little 
urea.  Such  observ-ations  suggest  that  possibly  a  small  part  of 
our  urea  may  come  from  uric  acid,  but  they  have  no  bearing 
on  the  proposition  that  the  acid  in  turn  has  its  origin  in  the 
nucleins  and  not  in  the  common  proteins. 

According  to  the  structural  formula  above  given  uric  acid 
appears  to  have  four  hydrogen  atoms  of  equal  value  in  the 
formation  of  salts.  But  apparently  only  two  classes  of  salts 
may  be  formed :  neutral  salts,  in  which  two  hydrogens  are 
replaced,  and  acid  salts,  in  which  but  one  hydrogen  is  replaced. 
We  have  therefore  salts  of  the  types  MC5H3N4O3  and 
AI2C5H2X4O;;.  In  addition  to  these,  so-called  quadriuratcs 
are  known  as  urine  sediments.  These  salts  are  of  the  type 
^1^1^3X403  •  C-,H4N403.  The  pure  acid  requires  nearly 
40,000  parts  of  water  for  solution ;  the  neutral  salts  of  the 
alkali  metals  are  much  more  soluble,  while  the  acid  salts  are 
but  slightly  soluble.  The  data  given  by  different  observers 
are  very  contradictor)^  The  salts  of  barium,  strontium  and 
magnesium  are  nearly  insoluble  in  water. 


THE    NITROGENOUS    EXCRETION.       URINE.  ^6^ 

Solutions  of  urates  in  presence  of  alkali  exhibit  a  reducing 
action  toward  copper,  silver  and  certain  other  salts,  which  fact 
possesses  an  importance  which  will  be  explained  later. 

XANTHINE  AND  THE  OTHER  PURINES. 

The  amount  of  these  purines  not  changed  to  uric  acid  is  small,  the  sum 
of  all  of  the  nitrogen  so  held  not  being  over  one-tenth  of  the  uric  acid 
nitrogen,  according  to  most  observers.  They  are  but  slightly  soluble  in 
pure  water,  but  dissolve  readily  with  alkali  hydroxides  to  form  compounds 
like  the  urates.  With  the  heavy  metals  the  combinations  are  mostly 
insoluble. 

PYRIMIDINE  DERIVATIVES. 

Among  the  decomposition  products  of  nucleinic  acids  the  so-called 
pyrimidines  should  be  referred  to,  as  they  must  bear  some  relation  to  the 
urinary  nitrogen.  Pyrimidine  itself  represents  a  nucleus  like  purine  from 
which  various  derivatives  may  be  formed.  The  relations  as  outlined  by 
Kossel  are  these : 

1  N=C— H  6  NH— C=0  NH— CO 

2  CH  C— H  5  C=0  C— H  C=0  C— CHj 

II      II  I  II  I  II 

3  N— C— H  4  NH— C— H  NH— CH 

Pyrimidine,  C^H^N,  Uracil,  C^HiN^Oa  Thymine,  CsHeN^O^ 

2,  6-dioxy  pyrimidine  5-metliyl  uracil 

We  have  no  definite  knowledge  of  the  occurrence  of  these  bodies  in 
urine,  but  they  are  among  the  compounds  which  should  be  expected  to 
result  from  the  nuclein  metabolism  and  possibly  form  a  part  of  the  nitro- 
gen residue  not  fully  accounted  for.  Thymine  has  been  obtained  as  a 
well-crystallized  compound,  soluble  readily  in  warm  water.  It  is  identical 
with  the  body  formerly  described  as  nucleosin. 

CREATININE. 

Creatinine,  C4H7N3O,  is  the  anhydride  of  the  creatine  de- 
scribed under  the  head  of  the  muscle  extractives,  and  is  always 
present  in  normal  urine.  The  amount  in  which  it  occurs  is 
indicated  by  the  analyses  given  above.  Much  has  been  writ- 
ten on  the  question  of  the  relation  of  creatine  to  urea,  but  the 
evidence  for  the  view  held  by  some  writers  that  the  latter  is 
derived  from  the  former  is  not  very  convincing.  In  a  labora- 
tory way  by  boiling  creatine  with  baryta  water,  urea,  sarkosine 
and  several  other  things  result.     But  no  corresponding  reac- 


368  PHYSIOLOGICAL    CHEMISTRY. 

tion  appears  to  take  place  in  transfusion  experiments  with 
creatine,  and  it  has  been  shown  that  creatine  introckiced  with 
the  food  appears  in  the  urine,  not  as  urea,  but  as  creatinine. 
On  the  other  hand,  it  is  possible  that  the  change  may  take  place 
in  the  muscles,  where  the  store  of  creatine  is  the  greatest,  and 
the  urea,  as  fast  as  formed,  may  pass  into  the  blood  to  be  later 
eliminated  in  the  urine,  while  the  other  residue  undergoes 
further  oxidation.  The  portion  of  the  muscular  creatine 
which  is  not  so  changed,  as  a  source  of  energy,  appears  in  the 
urine  as  creatinine  in  the  main.  In  presence  of  acids  a  mole- 
cule of  water  is  lost  and  the  anhydride  results : 

NH 
NH,  /  \ 

"     ^-C\     CH3  -  "     ^^-C\     CH,.CO  +  H,0 

^^CH^.COOH  CH3 

But  in  alkaline  solution  the  reverse  reaction  takes  place,  crea- 
tine being  formed.  Creatinine  may  be  separated  from  the 
urine  most  easily  with  zinc  chloride  as  a  crystalline  double  salt 
\vhich  is  yellow  because  of  the  coprecipitation  of  pigments ; 
when  purified  the  crystals  are  colorless.  Creatinine  is  found 
in  normal  amount  in  the  urine  of  vegetarians.  Toward  cop- 
per and  other  metallic  solutions  it  behaves  as  a  reducing  agent 
when  alkali  is  present. 

CARBOHYDRATES. 

Normally  no  large  amount  of  any  carbohydrate  passes  from 
the  blood  into  the  urine,  but  with  increased  sugar  concentra- 
tion in  the  blood  from  any  cause,  the  urine  may  contain  sugar 
in  more  than  traces.  When  sugars  are  consumed  in  more 
than  the  "  assimilation  limit  "  a  part  of  the  excess  may  be 
found  in  the  urine.  This  is  true  of  glucose  as  well  as  of  cane 
sugar  and  maltose. 

But  under  normal  conditions  there  is  evidence  that  traces 
of  several  carbohydrates  and  of  bodies  related  to  them  pass 
also  into  the  urine.  It  is  well  known  that  the  a-naphthol  reac- 
tion is  given  with  ordinary  normal  urines,  and  this  points  to 


THE    NITROGENOUS    EXCRETION.       URINE.  369 

some  carbohydrate  deriA-ative  which  can  yield  furfuraldehyde. 
The  other  dehcate  sugar  tests  give  the  same  indication.  Some 
of  the  carbohydrate  doubtless  appears  as  such,  but  a  portion 
of  it  probably  exists  in  the  form  of  glucoproteids  or  other 
complex  groups.  Part  of  the  carbohydrate  can  be  readily 
fermented,  while  another  portion  resists  the  action  of  yeast. 
Such  observations  may  be  interpreted  as  suggesting  the  pres- 
ence of  other  carbohydrates  than  the  common  glucose.  More 
will  be  said  about  this  below. 

Pathologically,  the  passage  of  even  large  quantities  of  sugar 
into  the  urine  is  a  common  phenomenon.  This  is  most  fre- 
quently observed  in  diabetes  mellitiis,  a  disease  in  which  the 
power  of  oxidizing  sugar  in  the  muscles  seems  to  be  wholly 
or  partly  lost.  It  has  been  already  mentioned  that  this  oxida- 
tion is  possibly  an  enzymic  operation  in  which  certain  muscle 
and  pancreas  enzymes  are  at  the  same  time  active.  In  this 
form  of  diabetes  several  hundred  grams  daily  of  sugar  may 
be  excreted. 

In  another,  "  artificial,"  form  of  diabetes  sugar  may  be 
caused  to  appear  in  considerable  quantity  temporarily  in  the 
urine.  The  administration  of  small  doses  of  phloridzin,  a 
glucoside,  is  followed  by  the  appearance  of  sugar  in  the  urine, 
but  this  condition  is  now  believed  to  depend  on  a  disturbance 
of  the  proper  functions  of  the  kidneys  rather  than  on  any  alter- 
ation of  the  sugar-oxidizing  power.  The  power  of  retaining 
the  sugar  in  the  blood  from  which  it  should  be  taken  up  and 
oxidized  by  the  muscular  tissues  depends  on  the  maintenance 
of  the  integrity  of  the  membranous  structures  of  the  kidneys. 
If  these  suffer  strain,  which  seems  to  be  the  case  after  admin- 
istration of  phloridzin,  a  little  sugar  more  than  the  normal 
may  be  able  to  diffuse  or  pass  through  in  some  manner.  It 
is  possible  that  many  other  substances  may  have  a  somewhat 
similar  action,  but  in  much  smaller  degree,  and  so  occasion 
sugar  excretions  still  within  what  may  be  called  "  normal  " 
limits. 


370  PHYSIOLOGICAL    CHEMISTRY. 

PROTEINS. 

As  in  the  case  of  the  sugars  so  with  the  proteins ;  there  is 
good  evidence  that  traces  of  these  nitrogen  compounds  are 
normally  present  in  the  urine.  The  amount  which  may  be  so 
present  is.  however,  very  small,  in  the  mean  not  over  40  to  50 
milligrams  per  liter.  With  these  traces  it  is  not  possible  to 
say  how  many  different  kinds  of  proteins  may  occur,  but  the 
serum  albumin  is  doubtless  the  most  abundant.  With  greatly 
increased  amounts  of  albumin  in  the  diet  the  excreted  albumin 
is  also  increased,  with  no  visible  impairment  of  the  kidney 
mechanism.  It  is  also  true  that  foreign  albumins  injected  into 
the  blood  circulation,  white  of  egg  for  example,  are  speedily 
eliminated  by  the  kidneys. 

Pathologically,  however,  the  serum  albumin  and  serum 
globulin  of  the  blood  may  pass  through  the  kidneys  in  consid- 
erable quantity.  This  is  occasionally  due  to  disturbances  in 
the  circulation  which  may  bring  about  an  increase  of  blood 
pressure,  but  ordinarily  is  due  to  structural  changes  in  the  kid- 
neys themselves,  through  which  the  power  of  perfectly  retain- 
ing albumins  of  the  blood  is  lost.  A  discussion  of  the  nature 
of  these  changes  is  not  within  the  scope  of  this  book,  and  for 
an  explanation  of  the  tests  which  are  employed  in  recognizing 
the  proteins  of  the  urine  the  reader  is  referred  to  works  on 
urine  analysis.  Many  of  these  tests  are  but  modifications  of 
the  delicate  protein  tests  described  in  one  of  the  earlier 
chapters. 

URINARY    SEDIMENTS. 

The  urine  when  passed  is  usually  clear,  but  frequently  it 
soon  becomes  cloudy  and  deposits  a  precipitate.  This  precipi- 
tate may  consist  of  a  variety  of  constituents  and  may  be  due 
to  several  causes.  Sooner  or  later  all  urines,  unless  specially 
protected  by  preservatives,  undergo  the  so-called  ammoniacal 
fermentation  in  which  urea  is  converted  into  ammonium  car- 
bonate by  one  of  several  bacteria.  When  this  alkaline  condi- 
tion is  reached  the  condition  of  equilibrium  in  which  the  vari- 
ous salts  exist  together  is  destroyed  and  insoluble  products  are 


THE    NITROGENOUS    EXCRETION.       URINE.  3/1 

commonly  formed  which  appear  as  precipitates.  The  alkaH- 
earth  phosphates  are  the  bodies  usually  thrown  out  in  this 
way. 

But  disturbances  in  the  equilibrium  may  result  from  changes 
of  temperature  also,  and  precipitation  occur  because  of  the 
simple  cooling  of  the  warm  voided  urine.  Many  of  the  pecu- 
liar urate  sediments  are  formed  in  this  way.  Sometimes  the 
separation  of  sediment  begins  in  the  bladder  or  ureters  and 
this  sediment  may  take  the  form  of  hard  concretions  or  calculi. 
In  years  past  numerous  attempts  have  been  made  to  explain 
the  formation  of  these  deposits,  especially  as  they  occur  within 
the  body.  The  theoretical  explanations  given  have  been  in 
general  far  from  satisfactory,  and  the  most  recent  studies  have 
only  gone  to  show  the  complexity  of  the  problem.  It  is  now 
coming  to  be  recognized  that  we  have  here  one  of  the  most 
difficult  problems  of  physical  chemistry,  which  like  other  ques- 
tions of  chemical  equilibrium  in  solution  may  be  approached 
only  through  elaborate  studies.  The  beginning  of  such  stud- 
ies may  be  seen  in  some  of  the  valuable  papers  which  have 
been  published  in  the  last  few  years  on  the  solubility  of  uric 
acid  and  its  salts,  and  the  degrees  of  dissociation  which  obtain 
in  the  various  solutions.  As  yet  these  matters  are  scarcely  in 
condition  for  elementary  presentation. 

The  recognition  of  the  general  character  of  the  sediments 
from  urine  is  most  readily  effected  by  the  aid  of  the  micro- 
scope, which  is  explained  in  text-books  of  urine  analysis. 

THE  REDUCING  POWER  OF  NORMAL  URINE. 

On  account  of  the  presence  of  some  of  the  bodies  described 
in  the  last  few  pages  normal  urine  exhibits  a  certain  reducing 
action  toward  metallic  solutions.  At  one  time  this  was 
ascribed  to  traces  of  carbohydrates  present,  but  later  doubt  was 
thrown  on  this  conclusion  and  the  presence  of  even  traces  of 
sugar-like  bodies  was  denied  by  most  writers  concerned  with 
the  question.  With  the  development  of  greater  accuracy  in 
the  methods  of  detecting  sugars,  especially  through  the  aid  of 


37^ 


PHYSIOLOGIC.\L    CHEMISTRY, 


the  pheiiylhydrazine  combination  and  the  formation  of  benzoic 
esters,  the  question  of  the  passage  of  traces  of  carbohydrates 
into  the  urine  seems  to  be  finally  settled  in  the  afiirmative,  but 
the  amount  of  sugar  which  may  be  so  identified  is  too  small 
to  account  for  the  total  reduction  easily  measured  by  copper 
solutions.  This  normal  reduction  cannot  be  quantitatively 
followed  with  the  ordinary  Fehling  solution,  but  if  a  dilute 
Pavy  solution,  as  described  by  the  writer  in  his  work  on  Urine 
Analysis,  is  used,  a  very  satisfactory  determination  may  be 
made.  This  modified  Pavy  solution  is  given  such  a  strength 
that  I  cc.  oxidizes  i  mg.  of  glucose  in  very  dilute  solution, 
approximating  0.2  per  cent  strength  or  less. 

In  a  large  number  of  tests  made  in  the  author's  laboratory 
a  few  years  ago  it  was  found  that  to  reduce  50  cc.  of  such  a 
solution  urine  volumes  ranging  between  14.9  cc.  and  58  cc. 
were  required.  The  mean  of  all  the  determinations  on  these 
normal  urines  was  23  cc.  In  hundreds  of  normal  urines  ex- 
amined since  similar  results  have  been  obtained.  In  all  these 
urines  the  creatinine  and  uric  acid  present  were  accurately 
determined  and  an  attempt  was  made  to  connect  these  bodies 
with  the  reduction. 

The  Reducing  Power  of  Creatinine.  The  reducing  power 
of  creatinine  may  be  easily  found  by  the  method  referred  to, 
using  the  weak  ammoniacal  copper  solution.  A  liter  of  this 
solution  contains  8.166  gm.  of  CuSO^  •  5H2O,  corresponding 
to  2.6042  gm.  of  CuO.  A  measured  volume  of  this,  usually 
25  or  50  cc,  is  reduced  by  a  creatinine  solution  of  definite 
strength  and  the  volume  required  noted.  The  details  of  some 
experiments  are  given  in  the  following  table.  The  copper 
solution  was  diluted  to  100  cc.  before  the  titration: 


Creatinine  |  Copper  Solution  i     CuO  Equiva-     Creatinine  Solu- 
in  100  cc.  '         Taken.                     lent.                  tion  Used. 

1 

Creatinine  to    \     ^ols.  Cup 

50  mg. 

50 
120 
120 

25  cc. 
25 
50 
50 

65. 1  mg. 

65.1 
130.2 
130.2 

92.5  cc. 
94.0 
76.0 
77.0 

92.5  mg.           1.998 
94.0                   1.967 
91.2          1         2.026 
92.4                   2.000 

Mean,                i  .998 

THE    NITROGENOUS    EXCRETION.       URINE. 


373 


It  appears,  therefore,  that  in  this  kind  of  solution  2  mole- 
cules of  copper  oxide  are  required  to  oxidize  i  molecule  of 
creatinine.  The  2  molecules  of  copper  oxide  yield  i  atom  of 
oxygen.  It  will  be  recalled  that  5  molecules  of  copper  oxide 
are  used  up  in  oxidizing  i  molecule  of  dextrose.  For  equal 
weights  the  reducing  power  of  dextrose  is  about  twice  as  great 
as  is  that  of  creatinine. 

Reducing  Power  of  Uric  Acid.  With  the  same  reagent 
the  reducing  power  of  uric  acid  may  be  measured,  the  uric  acid 
being  dissolved  with  a  little  alkali  to  form  a  soluble  urate. 
The  table  below  illustrates  the  relation  of  the  reducing  and 
oxidizing  solutions : 


Uric  Acid 
in  100  cc. 

Copper  Solution 
Taken. 

CuO  Equiva- 
lent. 

Uric  Acid  So- 
lution Used. 

Uric  Acid  to 
130.2  mg.  CuO. 

Mols.  CuO 

to  I  Mol. 

CBH^N^Oa. 

80  mg. 

80 
120 
120 

15.3  CC. 

25 
50 
25 

39.8  mg. 
65.1 
130.2 
65.1 

36     CC. 

58 

76.8 

37-8 

94. 2  mg. 
92.8 
91.8 
90.8 

2.92 
2.96 
2.99 
3-03 

Mean,                2.98 

From  this  it  appears  that  i  molecule  of  uric  acid  requires  3 
molecules  of  copper  oxide  for  oxidation  under  the  conditions 
of  the  test,  or  1.5  atoms  of  oxygen.  This  is  a  larger  amount 
of  oxygen  than  would  be  required  for  the  oxidation  of  uric 
acid  to  urea  and  alloxan.     This  requires  i  atom : 

C6H4N.O3  +  O  +  H2O  =  CON2H4  +  C4H2N2O4. 

But  secondary  reactions  also  take  place,  and  a  partial  oxida- 
tion to  parabanic  acid  may  be  represented  in  this  way : 

2C5H4N4O3  +  3O  +  2H.O  =  2CON.H4  4-  C4H2N2O4  +  CsH.N.Os  -f  CO2. 

This  possibly  represents  the  course  of  the  reaction  with  such 
a  solution. 

With  these  reducing  values  established  it  is  possible  to  esti- 
mate the  fraction  of  the  total  urine  reduction  which  is  not  due 
to  these  two  most  important  substances  besides  sugar.  To  do 
this  it  is  necessary  to  find  the  amount  of  uric  acid  and  creatinine 


374  PHYSIOLOGICAL    CHEMISTRY. 

in  a  given  volume  of  urine  and  determine  its  reducing  value 
in  terms  of  CuO  with  the  same  ammoniacal  solution.  If  the 
reducing  power  of  the  creatinine  and  uric  acid  be  subtracted 
from  the  total,  the  reduction  due  to  glucose  and  other  bodies 
is  arrived  at.  To  illustrate,  in  the  examination  of  a  large 
number  of  urines  these  values  were  found  : 

Total  reducing  power  of  i  cc.  of  urine  in  mg.  of  CuO  =  6.204 
Reducing  power  of  the  creatinine  present  in  same  terms  =  1.961 
Reducing  power  of  the  uric  acid  present  in  same  terms  ^  0.935 
Reducing  power  of  the  sum  of  uric  acid  and  creatinine    =  2.896 

Nearly  one-half  of  the  total  reduction  then  corresponds  to 
the  action  of  these  two  nitrogen  bodies,  while  the  reduction  of 
the  other  substances  is  equivalent  to  3.308  mg.  of  CuO  per  cc. 
If  this  is  calculated  as  glucose  it  represents  1.27  mg.  per  cc. 
As  several  other  bodies  contribute  to  this  reducing  action  the 
value  of  any  saccharine  substance  present  must  be  still  smaller. 
It  has  been  found  by  earlier  investigations  that  on  concentra- 
.tion  urine  loses  a  part  of  its  reducing  power.  This  suggests 
that  some  volatile  substances  may  be  responsible  for  part  of  it. 
It  should  be  mentioned  in  addition,  that  the  relative  extent  of 
the  reducing  action  varies  with  the  reagent  employed. 
Knapp's  mercury  solution  has  been  used  for  the  purpose  but 
it  is  not  as  convenient  as  the  ammoniacal  copper  solutions. 

A  knowledge  of  this  normal  reducing  action  is  not  without 
value,  since  very  frequently  the  mistake  has  been  made  of  as- 
suming the  presence  of  sugar  in  suspicious  quantities  in  the 
urine  merely  from  a  reduction  test.  In  some  of  the  extreme 
cases  quoted  in  one  of  the  statements  above,  the  normal  reduc- 
tion was  equivalent  to  over  0.3  per  cent  of  glucose,  when  in 
reality  it  was  largely  due  to  uric  acid  and  creatinine.  This 
point  has  importance  in  clinical  observations.  In  another 
direction  also  the  question  is  important,  as  the  reducing  power 
of  the  urine  is  a  measure  of  the  extent  of  certain  kinds  of  excre- 
tion. Creatinine  and  uric  acid  are  probably  perfectly  normal 
end  products  of  metabolism,  and  when  large  in  amount  the 
reduction  is  high.     It  will  be  recalled  further  that,  as  men- 


THE    NITROGENOUS    EXCRETION.       URINE.  3/5 

tioned  in  a  former  chapter,  strongly  reducing  substances  are 
produced  in  the  autolytic  changes  taking  place  in  the  liver 
and  pancreas.  In  cases  then,  this  "  normal  "  reduction  may 
become  excessive. 

THE  ELECTRICAL  CONDUCTIVITY  OF  URINE. 

In  one  of  the  chapters  on  the  blood  the  nature  of  electrical 
conductivity  in  the  fluids  of  the  body  was  explained  and  the 
methods  of  determination  outlined.  As  this  conductivity  de- 
pends mainly  on  the  sum  of  the  inorganic  constituents  present, 
and  as  sodium  chloride  is  the  most  abundant  of  these,  the  de- 
termination in  itself  has  but  a  limited  importance.  In  some 
cases  the  value  of  the  conductivity  would  be  merely  a  rough 
measure  of  the  salt  consumed  with  the  food,  and  the  salt  con- 
sumption is  extremely  variable. 

Nearly  all  the  other  substances  found  in  the  urine  have  a  sig- 
nificance very  different  from  that  of  the  salt.  The  latter  is  con- 
sumed and  excreted  as  such,  while  the  other  important  urinary 
constituents  are  products  of  metabolism,  that  is,  of  the  breaking 
down  of  the  digested  and  absorbed  food  materials.  The 
organic  products  of  metabolism  are  practically  non-electrolytes 
or  bodies  with  a  very  low  conducting  power;  indeed  the  con- 
ductivity of  a  weak  salt  solution  is  materially  lowered  by  the 
addition  of  urea  and  the  effect  of  the  purine  bodies  is  practi- 
cally in  the  same  direction.  Aside  from  the  chlorides,  the  in- 
organic salts  of  the  urine  are  mainly  phosphates  and  sulphates 
of  the  alkali  or  alkali-earth  metals,  and  these  are  made  up 
largely  from  the  oxidation  of  sulphur  and  phosphorus  of  pro- 
tein foods.  We  consume  a  certain  amount  of  phosphoric  and 
sulphuric  acids  in  complex  organic  combination,  in  the  lecithins 
and  chondroitins,  for  example,  and  small  amounts  of  mineral 
sulphates  and  phosphates  are  also  found  in  some  of  our  foods, 
but  these  amounts  are  not  large  enough  to  vitiate  the  truth  of 
the  general  proposition  that  the  sulphuric  and  phosphoric  acids 
as  detected  in  the  urine  are  results  of  certain  kinds  of  meta- 


376  PHYSIOLOGICAL    CHEMISTRY. 

holism.  Xow.  tlie  conductivity  measures  tlie  coml:)ined  effect 
of  these  products  of  oxidation  and.  if  it  could  be  determined 
apart  from  the  effect  of  the  chlorides,  a  factor  of  considerable 
practical  importance  would  be  secured. 

Approximately,  the  conductivity  due  to  metabolic  products 
may  be  found  by  subtracting  from  the  observed  conductivity 
that  due  to  sodium  chloride  in  the  same  solution.  The  chlorine 
may  be  accurately  determined  and  calculated  as  chloride;  then 
from  tables  the  conductivity  for  a  solution  of  this  concentration 
may  be  found  and  used  as  a  correction  to  be  taken  from  the 
total  observed  conductivity,  leaving  the  desired  residual  or 
metabolic  conductivity.  In  this  plan,  however,  an  error  is  in- 
volved, because  the  conductivity  of  the  chloride  taken  from 
the  tables  directly  is  that  found  in  pure  aqueous  solution  in 
absence  of  other  salts,  and  is  larger  than  the  true  conductivity 
of  the  chloride  as  it  exists  in  the  urine.  It  is  necessar}%  there- 
fore, to  use  as  a  correction  the  value  of  the  salt  conductivity, 
not  in  aqueous  solution,  but  in  a  solution  of  a  concentration 
corresponding  to  that  of  urine. 

Several  attempts  have  been  made  to  measure  the  conductivity 
of  mixtures  of  electrolytes  and  formulate  the  results.  Most  of 
the  experiments  have  been  made  with  dilute  solutions  and  can 
not  be  used  well  in  cases  like  the  present  one.  The  author  has 
investigated  the  question  with  special  reference  to  mixtures  like 
the  urine  and  has  considered  the  effect  of  urea  as  a  non-elec- 
trolyte also.  The  conductivity  varies  from  individual  to  in- 
dividual, and  from  hour  to  hour  according  to  the  kind  and 
amount  of  food  metabolized,  but  is  seldom  above  k  =  0.03. 
The  following  table  illustrates  the  variations  in  the  urine  of 
two  men.  both  well  nourished  on  mixed  diet,  with  the  water 
consumption  intentionally  high. 

By  making  complete  urine  analyses  and  duplicating  the  re- 
sults by  mixing  the  inorganic  and  organic  ions  and  the  non- 
electrolytes  in  proper  proportion,  it  is  possible  to  reach  almost 
exactly  the  corresponding  urine  conductivity  as  shown,  for  ex- 
ample, with  the  urines  the  complete  analyses  of  which  were 


THE    NITROGENOUS    EXCRETION.       URINE. 


377 


.2  c  o  o 

>  Z~,  o 
V  u  ^  t^ 


^E 


«     -2      " 

4^  S^  ^  ra   H 


rt  o       " 

<u  t-  3  \o 
XI  «.?  " 


X.2" " 


"■2 
o  S 

m"3 


A 

-o" 

>. 

VI 

> 

J^-o 

o°gK 

3 

"u   f 

O 

p. 

> 

U3 

14.5 

1.024 

178 

1.023 

13.S 

1.024 

118 

1.027 

128 

1.026 

458 

1,023 

6-9  A.M.  145  1.024  0.02589 

9-12  178  1.023  0.03021 

12-3  135  1.024  0.02834 

3-6  118  1.027  0.02790 

6-9  P.  M.  128  1.026  0.02694 

9-6  458  1.023  0.02421 

Means,  3-hr.  period  1.024  0.02650 


192 

1.020 

245 

1.020 

ISS 

1.026 

113 

1.026 

15s 

1.025 

405 

1.025 

6-9  A.  M. 
9-12 
12-3 

3-6 

6-9  P.  M. 

9-6 


Means,  3-hr.  period  1.024     0.02518 


6-9  A.  M. 
9-12 
12-3 
3-6 

6-9  P.  M. 
9-6 


0.02645 
0.02926 
0.02803 
0.02702 
0.02702 
0.02122 


230 

1.022 

260 

1. 021 

160 

1.026 

134 

1.026  1 

146 

1.025  1 

394 

1.023  1 

0.02683 

0.02939 

0.02792 

0.02755 

0.02580 
0.01997 


Means,  3-hr.  period  1.024      o.02/i 


112 

1.022 

220 

1. 019 

77 

1.026 

70 

1.027 

198 

1. 021 

184 

1.026 

0.02264 
0.02519 
0.02226 
0.02614 
0.02178 
0.02008 


1.024      0.02228 


410 

1.007 

0.01039 

122 

1. 017 

0.02408 

96 

1.023 

0.02422 

I3« 

1.024 

0.02355 

180 

1. 021 

0.02144 

285 

1.024 

0.02369 

1.020     0.02IJ 


278 

1. 015 

0.02629 

i5« 

1.022 

0.03037 

92 

1.029 

0.02720 

71 

1.030 

0.02566 

112 

1.027 

0.02577 

388 

1. 014 

0.01492 

1. 021      0.02251 


given  some  pages  back.     The  figures  here  given  show  the 
results : 


No.  of  Urine. 

I 

2 

3         4 

5         6 

K  as  observed. 
K.  from  mixtures. 

0.02372 
0.02332 

0.02402 
0.02400 

0.02793   0.01984 
0.02768   0.01944 

0.02898   0.02251 
0.02886   0.02158 

But  if  an  attempt  were  made  to  calculate  the  urine  conduc- 
tivity by  adding  together  the  individual  conductivities  of  the 
various  salts  for  the  concentrations  as  found  by  analysis  the 
result  would  be  too  high,  as  the  conductivity  of  each  substance 
is  lowered  somewhat  by  the  presence  of  the  others.     It  is  pos- 


^y8  PHYSIOLOGICAL    CHEMISTRY. 

sible,  however,  by  experimenting  with  known  artificial  mix- 
tures, to  determine  the  extent  of  this  modification  and  so  be 
able  to  introduce  a  correction.  For  the  present  purpose  the 
disturbing  action  of  the  other  urinary  constituents  on  sodium 
chloride  is  all  that  is  called  for.  This  has  been  done,  but  the 
details  of  the  investigation  can  not  be  given  here. 

In  a  long  series  of  experiments  it  was  found  that  a  mean 
correction  for  the  effect  of  sodium  chloride  may  be  made  in  this 
way.  The  chlorine  is  accurately  determined  in  the  urine  and 
calculated  as  sodium  chloride.  From  conductivity  tables  the 
value  of  K  for  the  calculated  concentration  is  found  and  this 
value  is  diminished  by  3  per  cent  which  is  the  average  correc- 
tion due  to  the  presence  of  other  substances,  organic  and  inor- 
ganic, in  the  urine.  This  corrected  salt  conductivity  in  turn 
must  be  subtracted  from  the  observed  total  conductivity  to 
find  the  fraction  due  to  metabolic  products.  This  correction 
gives  a  result  which  is  near  the  truth  in  ordinary  normal  urines 
and  which  may  be  taken  as  furnishing  a  peculiar  kind  of  meas- 
ure of  the  total  metabolism,  that  will  be  found  to  have  a  value 
in  certain  calculations. 

THE    FREEZING    POINT    OF    URINE.      CRYOSCOPY. 

In  the  thirteenth  chapter  the  application  of  cryoscopic 
methods  to  blood  examinations  was  discussed,  and  the  appa- 
ratus used  described.  In  urine  investigations  also  freezing 
point  determinations  have  become  important  and  a  very  con- 
siderable literature  has  accumulated.  The  Beckmann  appa- 
ratus may  be  employed,  as  with  the  blood,  but  the  Zikel  modi- 
fication has  been  found  to  give  good  results  and  is  somewhat 
simpler  in  manipulation. 

Conductivity  and  cryoscopic  methods  do  not  yield  exactly 
parallel  results;  the  conductivity  power  of  the  urine  depends 
essentially  on  the  number  of  inorganic  molecules  or  ions  pres- 
ent, while  the  freezing  point  depression  depends  on  the  sum 
of  all  the  dissolved  substances.     Urea  is  therefore  important 


THE    NITROGENOUS    EXCRETION.       URINE.  3/9 

in  the  one  instance,  but  not  in  the  other,  as  it  is  practically  a 
non-conductor.  This  being  the  case  it  is  evident  that  valuable 
information  may  be  obtained  by  a  combination  of  the  two 
methods,  as  it  is  possible  to  determine  the  fraction  of  the 
osmotic  pressure  of  the  urine  due  to  electrolytes  and  non-elec- 
trolytes.    Such  applications  are  frequently  made. 

While  the  osmotic  pressure  of  the  blood  is  nearly  constant, 
that  of  the  urine  is  extremely  variable.  Ordinarily  the  limits 
are  between  A  =  — 1.3°  and  — 2.0°,  but  after  great  water 
consumption  on  the  one  hand,  or  consumption  of  much  nitro- 
genous food,  or  salt,  without  sufficient  liquid,  on  the  other, 
the  freezing  point  of  the  urine  may  vary  from  A  ==  —  0.1° 
to  —  3-0°.  That  is,  the  urine  concentration  may  range  from 
one-fifth  that  of  the  blood  to  over  five  times  the  blood  concen- 
tration, expressed  in  active  molecules.  It  will  be  remembered 
that  a  freezing  point  depression  of  1°  C.  corresponds  to  an 
osmotic  pressure  of  12.  i  atmospheres. 

The  applications  of  this  cryoscopic  method  to  urine  are 
mainly  in  the  direction  of  diagnosis.  Since  it  is  possible  to 
collect  the  urine  from  each  kidney  separately,  by  ureter  cath- 
eterization or  equivalent  means,  a  test  of  the  two  portions  will 
disclose  any  difference  in  the  performance  of  the  two  organs. 
Normally,  the  secretions  from  the  two  kidneys,  for  a  given 
time,  should  be  the  same.  A  freezing  point  determination  is 
easily  made  and  will  show  if  one  kidney  is  doing  more  work 
than  the  other.  By  including  a  conductivity  test  it  may  be 
found  that  the  difficulty  in  excretion  is  more  pronounced  for 
one  class  of  substances  than  for  another. 


CHAPTER    XX. 

THE  GASEOUS  EXCRETION.     RESPIRATION. 

In  the  last  chapter  the  amount  of  nitroo^en  excreted  with  the 
urine  was  discussed  at  some  length.  With  the  nitrogen  certain 
corresponding  proportions  of  carbon,  hydrogen  and  oxygen  are 
excreted  in  the  urea,  uric  acid  and  other  bodies  described.  But 
the  larger  amounts  of  these  elements  are  thrown  off  from  the 
body  in  different  form,  and  especially  in  the  carbon  dioxide 
and  water  vapor  eliminated  in  respiration  and  perspiration. 
From  certain  classes  of  foods  the  end  products  formed  are 
these  two  only  when  the  oxidation  is  ideally  complete.  This 
is  the  case  with  the  fats  and  carbohydrates,  and  supposing 
them  wholly  burned  in  the  body  the  final  results  are  repre- 
sented in  this  way,  taking  typical  substances  for  illustration  : 

GHijOc  +  60=  =  6C0.  +  6H=0, 
C8H5(Ci8H350=)3  +  163O  =  57CO2  +  55H2O. 

In  the  actual  behavior  of  these  compounds  in  the  human 
body,  however,  the  results  are  somewhat  different.  The  oxi- 
dation is  never  quite  as  complete  as  here  indicated,  as  traces  of 
both  carbohydrates  and  fats  are  left  in  more  complex  forms. 

THE  RESPIRATORY   QUOTIENT. 

In  studying  the  completeness  of  oxidation  of  certain  foods 
much  has  been  learned  by  a  consideration  of  the  factor  known 
as  the  respiratory  quotient  which  is  simply  the  ratio  of  the 
carbon  dioxide  eliminated  to  the  oxygen  absorbed,  measured  by 
volume.  This  quotient  is  therefore  given  by  the  expression 
CO2/O2.  For  the  sugar  of  the  above  equation  we  require  six 
molecules  of  oxygen,  and  the  carbon  dioxide  produced  is  also 
six  molecules.     Hence  COo/Oa^i.     For  all  common  car- 

380 


THE    GASEOUS    EXCRETION.  38 1 

bohydrates  the  result  is  the  same.  For  the  fats,  however,  the 
quotient  is  much  smaller  since  57  COo  is  the  carbon  dioxide 
volume  excreted  for  an  oxygen  consumption  of  81.5  O2.  In 
this  case  002/02  =  57/81.5=0.7.  For  the  protein  bodies 
the  factor  cannot  be  as  easily  calculated,  since  we  are  not  able 
to  assign  a  formula  to  these  substances,  and  moreover  we  are 
not  familiar  with  all  their  oxidation  products.  But  from  the 
percentage  composition  and  the  known  facts  regarding  the 
elimination  of  urea,  uric  acid,  ammonia  and  creatine  it  is  pos- 
sible to  calculate  an  approximate  quotient.  This  is  about  0.8, 
which  factor  may  be  used  in  calculations. 

The  use  of  these  cjuotients  is  ordinarily  based  on  the  assump- 
tion that  the  oxidation  is  a  direct  one  and  that  corresponding 
to  the  oxygen  absorbed  there  is  almost  immediately  a  libera- 
tion of  carbon  dioxide  in  the  right  proportion.  But  this  as- 
sumption does  not  hold  absolutely  true ;  the  breakdown  of  car- 
bohydrate, for  example,  may  yield  at  first,  in  part,  products 
with  high  oxygen  content  from  which  COo  separates  later. 
In  other  words,  there  may  be  an  apparent  temporary  storing 
up  of  oxygen,  which  would  make  the  quotient  appear  low. 
Later  a  compensating  excessive  liberation  of  carbon  dioxide 
would  have  the  opposite  result.  However,  in  observations 
carried  out  through  a  period  of  proper  length  these  variations 
would  not  affect  the  general  mean. 

The  Carbon  and  Nitrogen  Balance.  The  body  is  in  car- 
bon equilibrium  when  just  as  much  carbon  is  eliminated  as  is 
consumed  in  the  food,  and  a  determination  of  this  element  in 
the  various  excreted  products  and  in  the  food  is  sufficient  to 
show  whether  there  is  gain,  loss  or  equilibrium  in  body  weight. 
All  the  food  stuffs  are  organic,  it  will  be  remembered,  and 
contain  carbon  as  the  fundamental  element. 

A  change  in  weight  may  result  from  gain  or  loss  in  fat  or 
gain  or  loss  in  protein.  Nitrogen  equilibrium  exists  when 
income  and  outgo  are  equal;  in  this  case  all  the  proteins  con- 
sumed as  foods  are  decomposed.  A  determination  of  nitrogen 
in  the  urine  and  feces  coupled  with  a  knowledge  of  the  food 


382  PHYSIOLOGICAL    CHEMISTRY. 

protein  will  decide  this  point,  since  the  excreted  nitroo-en  mul- 
tiplied bv  6.25  g-ives  a  measure  of  the  food  protein.  The  most 
accurate  method  of  reaching  the  value  of  the  excreted  nitrogen 
is  by  Kjeldahl  determinations  on  the  urine  and  feces,  but  good 
approximate  results  are  secured  by  determination  of  urea  alone, 
it  being  remembered  that  about  90  per  cent  of  the  urinary 
nitrogen  appears  in  this  form. 

Respiration  Apparatus.  To  determine  the  volume  of 
oxygen  inhaled  and  carton  dioxide  given  ofif  the  animal  or 
person  under  experiment  is  placed  in  a  respiration  chamber  of 
some  kind.  In  a  form  of  respiration  chamber  sometimes 
used,  an  accurately  measured  volume  of  air  with  known  con- 
tent of  moisture  and  carbon  dioxide  is  forced  through.  The 
air  leaving  the  tight  chamber  is  analyzed  and  the  amount  of 
carbon  dioxide,  moisture  and  oxygen  determined.  This  last 
determination  may  be  made  directly,  or  the  loss  of  oxygen  by 
the  respiration  of  the  person  in  the  cage  may  be  found  by 
calculation  from  this  basis :  The  sum  of  all  the  factors  con- 
sumed, that  is  the  food  and  the  oxygen,  plus  the  body  weight, 
must  be  l)alanced  bv  the  weight  of  the  body  at  the  end  of  the 
experiment  plus  the  various  excreted  matters.  If  A  repre- 
sents the  body  weight  at  the  beginning  of  the  test  and  A'  the 
body  weight  at  the  end  of  the  test.  Ox  the  oxygen  consumed, 
F  the  (dry)  food  consumed.  Ex  the  total  excreta  by  weight, 

then 

A-{-Ox^F  —  A'  +  Ex. 

Ox^A'  +  Ex—(A+F). 

In  some  of  the  recent  forms  of  respiration  apparatus,  espe- 
cially that  of  Atwater  and  Rosa,  extremely  accurate  results 
are  possible  in  the  determination  of  carbon  dioxide  and  mois- 
ture produced ;  but  with  increase  in  size  of  the  apparatus  a 
direct  determination  of  oxygen  difference  becomes  more  and 
more  difficult. 

In  the  Zuntz  apparatus,  which  is  often  used  for  short  experi- 
ments on  the  gaseous  excretion  only,  a  peculiar  mouthpiece  is 


THE    GASEOUS    EXCRETION.  383 

worn  which  permits  a  collection  of  the  carbon  dioxide  and 
vapor  from  the  lungs,  and  of  the  total  expired  air.  A  deter- 
mination of  the  oxygen  and  carbon  dioxide  is  accurately  made 
and  this  furnishes  all  the  data  necessary  for  the  calculation. 
The  nose  is  closed  in  this  experiment;  the  mouthpiece  is  so 
arranged  that  air  may  be  drawn  in  without  allowing  the  excre- 
tory products  to  escape. 

DEDUCTIONS  FROM  RESPIRATION  EXPERIMENTS. 

These  are  undertaken  to  answer  a  number  of  important 
questions.  The  weight  of  carbon  dioxide  excreted  may  reach 
fifteen  hundred  grams  or  more  daily  and  it  is  interesting  to 
know  under  what  circumstances  it  is  increased  and  when 
diminished.  Very  simple  observations  show  that  the  body  at 
rest  produces  much  less  of  the  gas  than  does  the  body  at  work. 
In  the  latter  condition  the  destruction  of  food  stuffs  is  called 
for  to  liberate  mechanical  energy.  This  is  practically  possible 
only  through  oxidation,  and  carbon  dioxide  is  the  first  tangible 
result  of  the  oxidation. 

The  question  also  comes  up,  what  kind  of  organic  matter 
is  most  readily  or  most  commonly  oxidized  when  work  is 
done?  On  this  question  much  has  been  written  and  our  views 
have  undergone  various  changes  through  the  years.  Liebig 
considered  the  proteins  as  the  foods  which  must  be  burned  to 
enable  us  to  do  mechanical  work,  but  in  a  famous  experiment 
by  Fick  and  Wislicenus,  undertaken  to  throw  some  light  on 
this  question,  no  great  excess  in  the  excretion  of  urea  was 
found  in  the  work  of  ascending  the  Faulhom,  and  the  protein 
oxidized  was  far  too  little  to  account  for  the  work  done. 
Other  investigators  reached  the  same  conclusion,  but  it  has 
been  found  that  under  certain  conditions  the  proteins  may  be 
consumed  to  do  work.  Ordinarily  fats  and  carbohydrates  are 
used  in  preference  and  no  large  amount  of  protein  is  used  if 
the  other  substances  are  present  in  sufficient  quantity. 

The  question  of  what  kind  of  foodstuff  is  oxidized  through 
periods  of  work  and  rest  may  be  answered  by  experiment.     As 


384  PHYSIOLOGICAL    CHEMISTRY. 

just  intimated,  examinations  of  the  urine  give  us  information 
as  to  the  nitrogen  excretion,  and  the  extent  of  oxidation  of  fats 
and  carbohydrates  may  be  measured  by  respiration  experi- 
ments. In  a  fasting  animal  at  rest  the  respiratory  quotient 
sinks  to  a  value  but  little  above  0.7,  showing  that  the  substance 
metabolized  is  mainly  fat;  as  some  proteins  are  also  used  up 
the  quotient  cannot  absolutely  reach  0.7.  If  work  is  done  by 
the  fasting  animal,  the  carbohydrate  bodies  of  the  muscular 
juices,  glycogen  essentially,  are  called  upon  and  their  effect  is 
added  to  that  of  the  proteins  in  raising  the  respiratory  quotient. 
On  the  other  hand,  it  has  been  found  that  a  well-fed  animal 
at  rest,  with  abundance  of  carboh3^drates  in  the  ration,  will 
excrete  a  volume  of  carbon  dioxide  nearly  as  great  as  that  of 
the  oxygen  absorbed.  In  this  case  the  ratio  CO2/O2  shows 
that  essentially  carbohydrates  are  burned  and  that  fat  is  al- 
lowed to  accumulate.  When  very  hard  work  is  done  by  the 
well-nourished  animal  the  quotient  sinks  to  an  intermediate 
value,  showing  that  fats  are  now  consumed  as  well.  This 
would  be  evident  also  from  observations  continued  over  a  long 
period  in  which  no  accumulation  of  fat  could  be  recorded. 
With  moderate  work  there  is  not  much  change. 

Some  of  these  results  are  illustrated  by  the  figures  in  the 
following  table  taken  from  observations  published  by  Chau- 
veau  and  Laulanie,  in  which  dogs  were  the  suljjects  of  experi- 
ment. These  figures  show  very  clearly  alteration  in  the  res- 
piratory quotient  with  work,  and  also  by  diet. 


Observed  Ratio  -~  ,  or  Respiratory  Quotient. 
0» 

^^              Food 

Consumption. 

1 

Before 
Work. 

Minutes  of  Work  Before 
Observations. 

Minutes  of  Rest  Follow- 
ing Work. 

30         45 

60 

90         120        180 

45         60     1    120 

240 

1  j  24  hours  fast. 

2  6  days  fast. 

3  I  day  fast. 

4  2  days  fast. 

5  3  days  fast. 

6  AfterfuUmeal. 

7  AfterfuUmeal. 

0.790 
0.750 
0.874 
0.740 
0.685 

1.033 
1. 000 

0.943 
0.819 

I.OI7 

0.905 
0.840 
0.895 
0.780 
0.790 

1.042 

0.900 

0.9000.900 
0.866  0.866 
0.8080.772 
1.044 

I  .C08 

0.789 

0.687 

0.7700.770 
0.7300.708 
0.681  0.681 
1.052 
1.032  1. 01 7 

0.756 

THE    GASEOUS    EXCRETION. 


385 


Other  experiments  are  in  general  good  agreement  with 
these.  The  effect  of  work  in  the  fasting  animal  is  seen  almost 
immediately.  In  the  last  experiments  the  respiratory  quotient 
is  greater  than  unity.  This  may  be  due  in  part  to  slight  errors 
in  observation,  but  it  should  be  remembered  that  there  are 
classes  of  compounds  in  which  such  a  result  would  always  fol- 
low. Such  compounds  are  not  common  articles  of  food,  but 
often  make  a  part  of  certain  vegetable  foods.  The  complete 
oxidation  of  tartaric  acid,  for  example,  would  yield  a  quotient 
of  1.6. 

The  quotient  may  sometimes  be  high,  as  intimated  above, 
if  the  oxygen  has  been  at  first  absorbed  to  form  compounds 
relatively  rich  in  oxygen,  which  are  later  broken  down  rapidly, 
under  working  or  other  conditions.  If  an  observation  is  made 
just  at  this  period  the  excess  of  CO2  liberated  would  present 
an  abnormal  result.  For  characteristic  results  the  observation 
periods  should  be  as  long  as  possible. 

Illustrative  Case.  Some  idea  of  the  importance  of  the 
respiratory  coefficient  determination  may  be  obtained  from  a 
consideration  of  the  following  assumed  case  in  which  the  con- 
ditions are  made  somewhat  ideal  for  simplicity  of  calculation. 
The  numerical  values  given  are  such  as  might  be  obtained  from 
the  mean  of  several  24-hour  experiments  in  a  large  respiration 
chamber.  The  diet  is  assumed  to  be  abundant  and  the  tests 
begun  after  a  condition  of  practical  nitrogen  equilibrium  is 
reached. 


Initial  weight   75  kilograms 

Final   weight    75-05  kilograms 


Income  Observed. 


^6 

a 

OCJ 

S3 

Pi 

G 
u 

V 
Oh 

^"3 
^e2 

0 

Total. 

H 

Total. 

Proteins 

Fats 

150 
no 

440 

35 
2,000 

53-5 
76.5 
44.2 

16.0 

23-5 
II. 4 
49.6 

7.0 
12. 1 

6.2 

80.3 
84.2 

194-5 

24.0 

35-2 

12.5 

218.2 

10.5 
^-3-3 

27-3 

Carbohydrates. 
Salts 

Water  

Total 

2,735 

359-0 

26 


386 


PHYSIOLOGICAL    CHEMISTRY. 


Outgo  Observed 

Weight  in 
Grams 

C. 

N. 

Salts. 

Vol.  CO.. 

Respiration,  CO„ 

Respiration,  H,0 

932 

904 

1.350 

74 

100 

33 

254.2 

30 
5 

471  1. 

Urine,  H,0 

8.7            20.4 

Urine,,  solids 

Feces,  H,0 

Feces,  solids 

16.2 

3-6 

Total 1     3,393      I 

279.1 

24.0 

35 

The  weight  of  the  various  excreted  products  is  greatly  in 
excess  of  the  visible  income,  but  the  oxygen  inhaled  has  not 
yet  been  calculated.  The  formula  given  above  may  be  applied 
to  find  this : 

Oxygen  =      A'      +      Excreta      —       {A       -f      F) 
75.050  3.393  75.000  2,735 

=  78,443  —  77,7ZS 

=  708  grams 
=  495.4  liters. 

In  the  table  above  the  volume  of  carbon  dioxide  eliminated  is 
given  as  471  liters.     The  respiratory  quotient  is  therefore 

^0= -47^  =3 .95. 
495-4 

This  gives  us  the  first  clue  as  to  the  nature  of  the  foods  metab- 
olized. The  factor  is  so  much  larger  than  that  correspond- 
ing to  the  fats  that  we  may  practically  exclude  these  at  once. 
In  any  event  there  is  a  large  protein  metabolism  since  the  orig- 
inal nitrogen  of  the  food  is  all  found  in  the  urine  and  the  feces. 
In  other  words  we  have  nitrogen  equilibrium,  with  no  storing 
up  of  protein  in  the  tissues.  The  respiratory  quotient  corre- 
sponds to  the  combustion  of  carbohydrates  and  proteins  mixed. 
If  we  assume  for  the  moment  that  no  fat  is  oxidized,  this 
calculation  may  be  made.  The  254.2  gm.  of  carbon  in  the  car- 
bon dioxide  of  respiration  calls  for  678  gm.  of  oxygen.  The 
difference  between  this  and  the  calculated  absorbed  oxygen, 
708  gm.,  amounts  to  30  gm.,  which  must  be  used  up  in  oxi- 
dizing hydrogen   of  protein   substances.     This   conclusion   is 


THE    GASEOUS    EXCRETION.  387 

drawn  because  the  carbohydrates  contain  enoug-h  oxygen  to 
burn  their  own  hydrogen,  and  the  protein  nitrogen  appears 
as  urea  and  calls  for  no  outside  oxygen.  The  burning  of  fat 
hydrogen  is  excluded  in  the  assumption. 

The  nitrogen  of  the  feces  corresponds  to  22.5  gm.  of  orig- 
inal protein  (6.25  X  3-6).  Not  all  of  this  nitrogen  is  actu- 
ally in  the  form  of  unchanged  or  residue  protein ;  a  part  of  it 
represents  products  of  metabolism  which  are  excreted  in  the 
feces,  as  explained  in  a  previous  chapter.  Probably  a  consid- 
erable fraction  may  be  considered  in  that  form;  but  it  must 
be  counted  as  a  loss  to  the  body,  and  we  have  therefore  as  net 
available  protein  (actually  used)  about  127.5  S^-  ^^  the 
final  metabolism  of  this  the  nitrogen  appears  in  urine  in  sev- 
eral forms,  but  mostly  as  urea.  The  per  cent  of  nitrogen  in 
this  is  46.7.  In  some  of  the  other  compounds  the  nitrogen 
is  higher  and  in  some  lower.  In  ammonia  much  hydrogen 
(relatively)  is  held,  and  in  uric  acid  little.  In  some  cases 
there  is  an  excess  of  carbon  and  in  other  cases  relatively  little 
carbon  is  held  with  the  same  weight  of  nitrogen.  The  various 
conditions  balance  each  other  pretty  well,  so  that  no  great 
error  will  be  made  if,  for  our  special  purpose,  we  count  all  the 
excreted  nitrogen  as  combined  in  the  form  of  urea.  We  have 
then  these  relations : 


C. 

N. 

H. 

0. 

In  metabolized  protein 

68.3 

8.7 

20.4 
20.4 

8.9 
2.9 

29.9 
II. 6 

In  urea 

59-6 

00.0 

6.0 

18.3 

To  oxidize  this  remaining  carbon  requires  159.8  gm.  of  oxy- 
gen. The  18.3  gm.  of  protein  oxygen  will  oxidize  about  2.3 
gm.  of  hydrogen.  The  remaining  hydrogen  from  the  6  gm. 
will  call  for  about  29.6  gm.  of  oxygen,  which  corresponds 
closely  to  the  amount  calculated  above. 

The  ingested  carbon  is  seen  from  the  table  to  be  359  gm. ; 
the  excreted  carbon  is  279  gm.,  from  which  it  follows  that  the 
body  has  gained  80  gm.     If  we  assume  this  to  be  in  the  form 


388  PHYSIOLOGICAL    CHEMISTRY. 

of  fat  the  latter  must  amount  to  about  104  gm.  This  repre- 
sents the  true  gain  in  body  weight ;  the  weighings  showed  a 
gain  of  only  50  gm.  The  discrepancy  may  be  accounted  for 
by  assuming  an  excessive  excretion  of  urine.  No  such  dis- 
crepancy would  appear  if  the  urine  were  passed  from  the  blad- 
der as  fast  as  formed,  but  as  it  is  collected  at  intervals  it  is 
not  possible  to  obtain  exactly  comparable  results. 

In  the  feces  there  must  be  some  carbon  deri\-ed  from  fats ; 
but  the  amount  cannot  be  large,  because  for  the  nitrogen  of  the 
feces  we  must  calculate  at  least  10  or  12  gm.  to  correspond. 
This  would  leave  about  5  gm.  of  carl^on  from  other  sources, 
accounting  for  the  discrepancy  between  consumed  fat  and 
de^Ktsited  fat. 

The  above  calculations  illustrate  the  principles  involved ;  in  an  actual 
practical  observation  the  method  would  be  the  same,  but  the  interpretation 
of  results  might  not  be  as  simple,  especially  with  a  low  respiratory  quotient 
found.  In  the  above  tables  the  salts  taken  with  the  food  are  assumed  to 
include  those  to  be  formed  by  the  oxidation  of  the  protein,  and  the  latter 
substance  figured  as  income  is  assumed  to  consist  of  the  organic  elements 
only.  A  slight  error  is  introduced  in  the  calculation  in  this  way,  but  that 
IS  not  considered.  In  actual  practice,  of  the  carbohydrates  some  little 
would  escape  complete  metabolism.  The  above  results  would  correspond 
to  a  completely  burned  carbohydrate. 

In  the  above  table  of  observations  the  total  oxygen  in  the  consumed 
substances,  including  the  water,  is  2,044  g"i-  In  the  excreted  products, 
allowing  30  gm.  for  the  solids  of  the  urine  and  feces  coming  from  bodies 
other  than  the  original  salts,  the  oxygen  appears  to  amount  to  about  2,800 
gm.  The  difference  shows  an  excess  of  756  gm.  while  the  calculation 
above  gave  708  gm.  of  oxygen  taken  in.  The  discrepancy  is  due  to  the 
excess  of  water  excreted  as  urine.  It  will  be  noticed  also  that  there  is 
a  great  excess  of  excreted  water  over  the  2,000  gm.  consumed.  This 
amounts  to  over  350  gm.,  of  which  300  gm.  would  come  from  the  com- 
bustion of  the  carbohydrates  and  proteins  metabolized. 

SKIN  RESPIRATION, 

It  is  usually  assumed  that  the  gaseous  exchange  is  wholly 
through  the  lungs,  but  this  is  not  quite  correct.  Experiments 
with  men  and  animals  have  shown  an  absorption  of  oxygen 
and  an  escape  of  carbon  dioxide  through  the  skin.  A  num- 
ber of  observers  have  put  results  for  the  latter  on  record  which. 


THE    GASEOUS    EXCRETION.  389 

however,  are  not  in  good  agreement.  For  1.6  square  meters 
of  skin  surface  the  resuhs  found  in  seven  observations  varied 
from  2.2  gm.  to  32  gm.  in  24  hours.  The  last  resuh  is  prob- 
ably much  too  high.  It  has  been  noticed  further  that  the 
amount  of  carbon  dioxide  escaping  through  the  skin  is  in- 
creased greatly  by  temperature.  The  excretion  at  30°  seems 
to  be  several  times  as  great  as  at  20°. 

For  the  absorption  of  oxygen  no  exact  figures  are  given,  but 
the  amount  is  very  small.  In  some  of  the  lower  animals,  how- 
ever, a  large  part  of  the  absorbed  oxygen,  as  well  as  of  the 
excluded  carbon  dioxide,  may  be  by  way  of  the  skin.  This 
has  been  shown  especially  in  the  frog,  where  after  removal  of 
the  lungs  a  nearly  normal  exchange  may  be  noted  for  a  period 
of  days. 

The  question  of  the  excretion  of  other  gases  than  carbon 
dioxide  by  the  skin,  and  the  lungs  also,  has  been  much  dis- 
cussed. Formerly  it  was  held  that  a  very  appreciable  quan- 
tity of  organic  gaseous  bodies  is  given  off  through  the  skin 
and  this  elimination  was  considered  necessary  for  the  well 
being  of  the  body.  The  unpleasant  odor  of  the  air  of  a 
crowded  room  was  ascribed  to  these  organic  emanations.  But 
much  doubt  has  been  thrown  on  this  notion  by  various  experi- 
ments, some  of  which  are  of  very  recent  date,  which  seem  to 
show  that  these  odors  come  not  through  the  skin  but  from 
decaying  substances  on  the  surface  of  the  skin  or  from  the 
clothing,  if  it  is  old  and  soiled.  Experiments  have  been  made 
of  testing  the  air  drawn  through  a  small  respiration  chamber, 
enclosing  the  body  of  a  man  to  the  neck,  with  perfectly  clean 
skin  and  clothed  in  fresh,  clean  garments.  Such  air  is  prac- 
tically without  odor  and  has  no  action  on  solutions  of  perman- 
ganate through  which  it  is  aspirated.  It  is  free  from  ammo- 
nia. The  odors  of  perspiration  are  apparently  largely  due  to 
the  fermentation  changes  of  solid  or  semi-solid  substances  on 
the  surface  of  the  skin  rather  than  to  excreted  gaseous  prod- 
ucts passing  through  the  pores  with  the  water.  It  has  been 
found  also  that  the  whole  surface  of  the  body  may  be  covered 


390  PHYSIOLOGICAL    CHEMISTRY. 

with  varnish  witliout  hamiful  result  if  precautions  are  taken 
to  prevent  loss  of  heat. 

TIME  AND   PLACE  OF  OXIDATION. 

The  determination  of  the  respiratory  quotient  through  short 
intervals  shows  considerable  variations,  as  pointed  out  some 
pages  back.  The  human  organism  has  not  the  power  of  stor- 
ing up  oxygen  in  the  free  or  combined  form  through  a  long 
period,  as  appears  to  be  the  case  with  some  cold-blooded  ani- 
mals, which  are  able  to  exist  for  a  time  in  an  atmosphere  free 
from  oxygen.  \\'ith  man  and  warm-blooded  animals  in  gen- 
eral this  is  not  possible;  with  these  life  without  oxygen  may 
be  maintained  for  but  a  few  minutes  at  most.  An  exception 
exists  in  the  case  of  those  animals  which  pass  the  winter  in  a 
dormant  condition  (hibernating  animals)  and  for  human 
beings  in  trance.  Here  the  absorption  of  oxygen  and  excre- 
tion of  carbon  dioxide  are  reduced  to  a  minimum.  But  ordi- 
narily man  and  the  higher  animals  require  some  inflow  of 
oxygen  all  the  time. 

The  extent  to  which  this  oxygen  is  used  depends  on  the 
activity  of  the  muscles  largely.  In  rest  periods  the  amount 
of  oxygen  taken  up  by  the  muscles  is  much  greater  than  is  the 
carbon  dioxide  given  off,  but  with  the  contracting  or  working 
muscle  the  reverse  is  the  case.  In  experiments  in  which  the 
changes  in  the  blood  supply  of  individual  muscles  may  be  fol- 
lowed it  may  be  shown  that  for  rest  periods  the  respiratory 
quotient  for  the  muscle  may  fall  far  below  0.7  or  even  below 
0.5.  From  the  rapidly  contracting  muscle,  on  the  other  hand, 
the  evolution  of  carbon  dioxide  is  relatively  great.  A  respira- 
tory quotient,  for  the  muscle,  of  1.5  or  even  2  or  more  may  be 
found.  This  indicates  that  during  rest  oxygen  may  be  taken 
up  from  the  blood  and  held  or  condensed  in  some  manner  by 
substances  within  the  muscular  tissue,  but  of  the  mechanism 
of  this  reaction  unfortunately  but  little  is  known.  In  doing 
work  tissue  is  rapidly  oxidized  at  the  expense  of  the  stored-up 
oxygen,  and  a  great  excess  of  carbon  dioxide  is  given   ofif 


THE    GASEOUS    EXCRETION.  391 

quickly.  These  changes  follow  one  upon  the  other  rather 
rapidly.  In  the  oxygen-absorbing  stage  some  intermediate 
products  are  probably  built  up  from  sugar  or  glycogen  or  other 
substances,  which  fall  apart  with  liberation  of  water  and  car- 
bon dioxide  in  the  succeeding  active  condition  of  the  muscle. 
The  problem  of  oxidation  in  the  tissues  is  possibly  somewhat 
like  that  of  etherification  in  which  alcohol  yields  ether  indi- 
rectly through  the  intermediate  ethyl  sulphuric  acid,  and  sev- 
eral suggestions  have  been  brought  forward  as  to  the  character 
of  complexes  formed  in  one  stage  of  the  oxidative  metabolism 
to  be  decomposed  in  another.  At  the  present  time  these  sug- 
gestions are  practically  wholly  within  the  realm  of  speculation, 
and  not  therefore  suitable  for  presentation  in  this  place.  It 
is  likely  that  all  these  reactions,  which  seem  to  be  carried  on 
in  the  tissues  rather  than  in  the  fluids  of  the  body,  are  incited 
by  enzymic  ferments,  and  to-day  certain  classes  of  oxidases 
are  often  assumed  to  be  the  agents  active  in  the  changes.  For 
some  of  the  oxidations  it  has  been  pretty  well  settled  that  an 
enzyme  produced  by  the  pancreas  is  necessary. 


CHAPTER    XXI. 
THE  ENERGY  EQUATION. 

We  come  now  to  a  brief  consideration  of  one  of  the  most 
important  questions  connected  with  the  whole  animal  chem- 
istry and  this  is  the  question  of  the  liberation  of  energy  from 
the  consumption  of  various  foods.  The  function  of  the  food 
we  eat  is  a  multiple  one.  It  may  not  only  increase  the  body 
weight  and  maintain  the  various  functions  of  the  body  through 
oxidation,  but  in  its  combustion  heat  is  liberated  to  maintain 
also  the  body  temperature,  and  energy  is  furnished  to  enable 
us  to  perform  external  work.  It  is  interesting  to  measure  the 
effect  of  the  food  in  these  several  directions,  which  may  be 
done  with  a  fair  degree  of  accuracy.  The  following  consid- 
erations will  show  the  basis  of  the  calculations. 

POTENTIAL  ENERGY  OF  FOOD. 

The  food  consisting  essentially  of  combustible  substances  is 
the  source  of  a  large  amount  of  potential  energy.  In  the  com- 
plete combustion  of  the  fats,  carbohydrates  and  proteins  of 
the  food  a  large  amount  of  heat  is  liberated  and  this  in  turn 
is  the  equivalent  of  a  certain  amount  of  work.  The  potential 
energy^  of  chemical  substances  may  be  measured  in  various 
ways,  but  for  purposes  like  the  present  it  is  customary  to  meas- 
ure this  energy  in  terms  of  the  units  of  heat  liberated  in  the 
combustion  of  the  body  in  question  with  oxygen.  Certain 
units  are  in  common  use : 

Unit  of  Heat,  Calorie.  The  unit  of  heat  or  calorie  may 
be  defined  as  the  quantity  of  heat  required  to  raise  the  tem- 
perature of  a  gram  of  water  one  centigrade  degree,  at  a  mean 
temperature.  As  the  heat  absorption  of  a  gram  of  water  is 
not  quite  the  same  throughout  the  scale,  the  calorie  is  perhaps 

392 


THE    ENERGY    EQUATION.  393 

more  satisfactorily  defined  as  the  one  hundredth  part  of  the 
quantity  of  heat  required  to  raise  the  temperature  of  a  gram 
of  water  from  o°  to  ioo°  C.  This  gives  the  ordinary  or  small 
calorie.  In  deahng  with  large  heat  transfers  a  larger  unit  is 
preferable  and  one  just  looo  times  as  large  is  frequently  used. 
In  this  the  kilogram  in  place  of  the  gram  of  water  is  warmed, 
and  the  unit  is  called  the  large  calorie.  The  first  may  be  ab- 
breviated cal.  and  the  second  Cal. 

Unit  of  Work  and  Unit  of  Force.  The  unit  of  force  is 
called  the  dyne  and  may  be  defined  as  the  force  which,  acting 
for  I  second  on  a  mass  of  i  gram,  gives  to  it  an  acceleration 
of  I  centimeter  per  second.  The  force  of  gravity  at  the  sea 
level  is  about  981  dynes,  since  this  adds  to  a  falling  body  an 
acceleration  of  981  cm.  per  second. 

The  unit  of  zfork  is  the  erg,  and  it  may  be  defined  as  the 
work  done  in  overcoming  unit  force  through  unit  distance. 
One  dyne  acting  through  one  centimeter  gives  us  one  erg  of 
work.  To  lift  i  gram  through  i  centimeter  requires  981  ergs 
of  work. 

Mechanical  Equivalent  of  Heat.  Work  may  be  done  by 
the  proper  utilization  of  heat,  and  in  turn  work  may  be  wholly 
converted  into  heat.  It  is  possible  therefore  to  express  one 
m  terms  of  the  other.  The  mechanical  or  work  equivalent 
of  a  unit  of  heat  has  been  determined  many  times  by  very 
elaborate  experiments.  If  a  given  quantity  of  heat  could  be 
applied  wholly  to  the  lifting -of  a  weight  it  would  be  found,  in 
accordance  with  the  mean  results  of  these  experiments,  that  i 
calorie  would  be  able  to  lift  423.5  gm.  through  i  meter,  or  i 
gm.  of  substance  through  423.5  meters.  Conversely,  if  a 
gram  of  water  be  dropped  from  a  height  of  423.5  meters,  and 
its  energy  of  motion  wholly  converted  into  heat,  its  tempera- 
ture will  be  found  to  be  increased  i  °  C.  We  have  then  these 
relations : 

I  calorie  :=  42,350  gm.  cm. 
=  41,500,000  ergs. 

Heats  of  Combustion.     By  means  of  calorimeter  expe^i- 


394 


PHYSIOLOGICAL    CHEMISTRY. 


ments  the  following  heats  of  combustion  have  been  determined. 
Results  found  by  different  workers  show  slight  variations,  but 
the  values  here  are  mean  values  and  sufficient  for  illustration. 


Fig.  31.  Bomb  calorimeter  (^Hempel-Atwater)  in  which  heats  of  combus- 
tion of  food  stuffs  are  determined  by  burning  in  oxygen.  A  is  the  bomb 
proper,  inside  of  which  a  small  platinum  capsule  is  shown  in  which  the  sub> 
stance  is  contained,  and  ignited  by  means  of  a  current  led  in  through  the  in- 
sulated wires.  The  bomb  is  immersed  in  water  ;  the  increase  in  temperature 
is  measured  by  means  of  a  delicate  thermometer. 


THE    ENERGY    EQUATION. 


395 


The  number  of  calories  furnished  by  burning  i  gm.  of  sub- 
stance in  each  case  is  given. 


Table  of  Heats  of  Combustion. 


Hydrogen   34,200 

Carbon 8,ioo 

Ethyl   alcohol    7,060 

Glycerol    4,200 

Mannitol  4,000 

Palmitic  acid    9,300 

Stearic  acid  9,400 

Fats,   average    9,400 

Hexoses    3,7oo 


Cane  sugar   4,000 

Starch    4,200 

Casein 5,700 

Egg  albumin  5,700 

Urea 2,500 

Uric  acid    2,700 

Leucine   6,500 

Tyrosine 6,000 

Creatine,  anhyd    4,250 


These  values  are  for  complete  combustion,  but  as  the  proteins 
in  the  body  are  not  oxidized  to  leave  water,  carbon  dioxide 
and  nitrogen,  we  must  subtract  from  the  given  values  the 
heats  of  combustion  of  the  urea,  uric  acid,  creatine  and  other 
products  found  in  the  urine,  in  order  to  secure  the  physiolog- 
ical heats  of  combustion  with  which  we  are  practically  con- 
cerned. 

DISTRIBUTION   OF  FOOD   ENERGY. 

With  these  preliminary  considerations  we  are  able  to  look 
at  the  manner  in  which  the  energy  of  the  consumed  food  is 
distributed.  On  the  one  side  we  have  the  substance  burned, 
on  the  other  the  products,  which  may  be  represented  diagram- 
matically  in  this  way : 


Potential  energy  of 

Food. 


Potential  energy  of 

Flesh  gained. 
Feces. 
Urine. 
Perspiration. 
Kinetic  energy  of 

Work. 
Heat. 

Experimentally,  the  whole  of  the  kinetic  energy  may  be 
made  to  take  the  form  of  heat,  which  simplifies  the  observa- 
tions materially.  It  is  practically  possible  to  determine  the 
heat  liberation  in  the  large  respiration  calorimeters  already  re- 
ferred to,  and  the  use  of  such  apparatus  will  be  explained 


396  PHYSIOLOGICAL    CHEMISTRY. 

below.  First,  however,  a  general  method  of  calculating  the 
energy  liberated  as  heat  will  be  given. 

CALCULATION   OF   KINETIC   ENERGY   OF   FOOD. 

In  illustration  of  this  we  may  make  use  of  the  example  given 
in  the  last  chapter  and  employ  a  method  which  in  principle  is 
very  simple.  The  income  of  energy  is  due  to  the  consumption 
of  certain  weights  of  protein,  fat  and  carbohydrates,  the  last 
of  which  we  may  assume  is  made  up  of  9  parts  of  starch  and 
I  part  of  cane  sugar,  all  weights  referring  to  the  anhydrous 
condition.  The  effect  of  the  oxidation  of  sulphur  and  phos- 
phorus will  be  neglected  here,  and  the  protein  will  be  assumed 
pure  carbon,  hydrogen,  oxygen  and  nitrogen.  We  have  then 
as  income: 

From  150  gm.  protein,  150X5700=    855,000 

no  gm.  fat,  no  X  9400  =  1,034,000 

440  gm.  carbohydrate.    440X4180=1,839,200 

3.728.200 

In  small  calories  the  whole  income  is  therefore  equivalent  to 
3.728,200  cal. 

We  have  next  to  calculate  the  potential  energy  of  the  food 
stuffs  not  actually  consumed,  which  are  left  in  the  feces  and 
the  urine,  and  also  the  energy  of  any  substance  which  may  be 
put  down  as  a  gain  in  weight  in  the  body.  Recalling  the  data 
of  the  experiment  in  the  last  chapter  we  have 

^33  gm-  feces  with  16.2  gm.  C. 
1,424  gm.  urine  with  20.4  gm.  N. 

Calculating  the  X  of  the  urine  as  urea,  which  in  practice  would 
not  be  quite  accurate,  we  have  44  gm.  of  that  substance.  The 
organic  matter  of  the  feces  corresponds  approximately  to  22.5 
gm.  of  bodies  resembling  protein  and  5.5  gm.  of  bodies  resem- 
bling fats,  and  these  data  we  can  now  employ  in  the  calcu- 
lation. 

The  illustration  gave  also  a  gain  of  80  gm.  of  fat.  The 
solid  matter  lost  in  the  form  of  perspiration  is  so  small  that 


THE    ENERGY    EQUATION.  397 

it  may  be  ignored  for  the  present  purpose.     We  have  then  the 
following  deductions  to  make : 

Potential  energ}'  in      80  gm.  of  fat  stored    752,000 

^33  gm.  of  feces    180,000 

1,424  gm.  of  urine    110,000 

1,042,000 

This  leaA^es  as  a  balance  to  be  calculated  as  kinetic  energ}- 

3,728,200 
1,042,000 

2,686,200  calories 

Another  method  of  calculation  deals  with  the  carbon  and  hydrogen  of  the 
food  and  feces  only.  The  heat  production  was  at  one  time  assumed  to  de- 
pend on  the  combustion  of  the  carbon  of  the  fats,  carbohydrates  and  pro- 
teins and  the  hydrogen  of  the  fats  and  proteins.  The  hydrogen  of  the  car- 
bohydrates was  not  considered  because  it  was  supposed  to  be  closely  com- 
bined with  the  oxygen  present  in  the  same  compounds  in  such  a  form  as 
to  yield  no  more  heat  on  oxidation.  In  like  manner  the  combustion  heats 
of  the  urine  and  feces  may  be  calculated  from  the  whole  carbon  and  hydro- 
gen content  of  the  organic  substances.  The  total  carbon  of  the  food  in  the 
experiment  is  395  gm.,  of  the  hydrogen  in  fats  and  proteins  23.8  gm.  The 
carbon  of  the  urine  and  feces  is  24.9  gm.,  while  the  hydrogen  of  the  urine 
and  feces  is  about  5.2  gm.     We  have  then : 

Heat  units  from  359  gm.  of  food  carbon 2,907,900 

Heat  units  from  23.8  gm.  of  food  hydrogen 813,900 

3,721,800      3,721,800 

Heat  units  from  24.9  gm.  of  excreta  carbon 201,690 

Heat  units  from    5.2  gm.  of  excreta  hydrogen. .  .  .      177,840 

379.-530         379,530 

Net  calories   3,342,270 

From  this  result  the  value  of  the  energy  stored  as  fat  would  have  to  be 
subtracted  as  before.  This  method  of  calculation  gives  a  somewhat  lower 
result  than  the  other,  and  largely  because  of  the  uncertainty  in  allowing 
for  the  excreted  carbon  and  hydrogen,  but  it  has  value  as  a  comparison 
process. 

Respiration  Calorimeters.  In  experiments  with  men  or  large  animals  on 
the  combustion  of  food  and  liberation  of  heat  some  kind  of  respiration  ap- 
paratus is  employed.  Some  modification  of  a  type  originally  introduced 
by  Pettenkofer  is  generally  used.  In  this  the  subject  is  placed  in  a  cham- 
ber with  double  walls  through  which  a  current  of  air  may  be  forced  and 


398 


PHYSIOLOGICAL    CHEMISTRY. 


iinifornily  mixed  inside.  A  known  part  of  the  ingoing  air  and  of  the  ont- 
going  air  may  be  diverted  for  analysis  so  as  to  permit  an  exact  determi- 
nation of  the  amount  of  oxidation  products  liberated  at  any  time.     The 


Atwater  and  Rosa  calorimeter  is  the  most  complete  of  all  such  construc- 
tions. In  this  the  heat  liberated  by  the  subject  is  taken  up  by  a  current 
of    cold    water    circulating    through    numerous    coils    of    pipe    inside    the 


THE    ENERGY    EQUATION.  399 

chamber  and  in  such  a  way  as  to  maintain  a  perfectly  uniform  temperature 
in  the  chamber  space.  The  walls  of  the  chamber  are  made  of  compart- 
ments containing  two  layers  of  air  and  two  layers  of  water  maintained  in 
such  relations  that  they  prevent  gain  or  loss  of  heat.  The  whole  heat 
liberation  is  taken  up  by  the  circulating  water  and  may  be  accurately 
measured.  The  respiration  chamber  has  a  capacity  of  about  175  cubic 
feet  and  is  large  enough  to  contain  a  chair  and  small  table  for  the  con- 
venience of  the  occupant  and  a  couch  to  sleep  on  at  night.  The  construc- 
tion is  such  that  food  may  be  passed  in  and  the  urine  and  feces  removed 
without  making  any  appreciable  change  in  the  temperature  or  content  of 
the  air  inside.  With  such  an  apparatus  it  is  possible  to  carry  on  a  test  of 
many  days  duration  and  obtain  extremely  accurate  and  important  results. 

In  work  experiments  in  such  a  calorimeter  a  bicycle  is  mounted  so  that 
work  is  done  against  friction.  The  final  effect  is  increased  heat  liberation, 
measured  as  before. 

The  construction  of  this  large  calorimeter  suggested  the  building  of 
still  larger  ones  of  the  same  general  type.  Some  of  these  are  being  used 
in  agricultural  experiment  stations  in  metabolism  experiments  on  large 
animals,  from  which  results  of  great  practical  value  may  be  expected. 

DISTRIBUTION  OF  THE  HEAT  ENERGY. 

According  to  the  first  calculation  above  we  have  from  the 
700  grams  of  food  consumed  a  balance  of  2,686,200  calories. 
It  remains  to  show  about  how  this  may  be  dissipated.  If 
retained  in  the  body  it  would  soon  bring  the  latter  to  the  boil- 
ing point.  But  the  heat  liberated  in  the  combustion  of  the 
food  finds  several  outlets,  the  most  important  of  which  will 
be  now  indicated.  In  the  first  place  the  urine  and  feces  leave 
the  body  at  a  temperature  much  higher  than  that  of  the  water 
consumed;  the  water  of  respiration  and  perspiration  has  to 
be  vaporized  at  the  expense  of  heat ;  the  air  inhaled  is  warmed 
to  a  temperature  of  37°,  which  is  in  the  mean  20°  higher  than 
when  taken  in.  The  specific  heat  of  the  air  (at  constant  pres- 
sure) is  about  0.25.  We  have  then,  approximately,  the  fol- 
lowing relations,  assuming  15  kilograms  of  air  to  be  inhaled 
in  the  24  hours  : 

To  warm  15.000  gm.  air  20° 75>ooo  cal. 

To  warm  1,557  gn^-  urine  and  feces  20° 31,140 

To  evaporate  904  gm.   of  water    (904  X  580) 524,320 

630,460 


400  PHYSIOLOGICAL    CHEMISTRY. 

This  number  of  calories  must  be  taken  from  the  net  produced 
calories  to  obtain  the  heat  radiated  or  otherwise  lost  by  the 
body.     \\'e  ha\e  then  this  difference : 

2,686.200 
630,460 

2,055,740 

That  is.  something  over  2,000,000  calories  are  dissipated  by 
radiation. 

HEAT    RADIATION    WHEN    WORK   IS    DONE. 

All  these  calculations  are  based  on  the  assumption  that  no 
mechanical  work  is  being  done  by  the  person  under  observa- 
tion, or  if  done  it  is  finally  all  converted  into  heat.  In  experi- 
ments in  the  respiration  calorimeter  a  very  close  agreement 
is  found  between  the  calculated  and  observed  heat  or  energy 
liberations.  This  is  illustrated  by  the  results  of  one  of  the 
Atwater  and  Rosa  experiments.  The  figures  are  the  daily 
means  from  tests  running  through  4  days : 

Total  energy  of  food,  determined 3,6/8  Cal. 

Energy  of  urine  and  feces 264 

Net    energ}' 3,414 

Energy  of  fat  lost 488 

3,902 
Energy  stored  as  protein 38 

Total  energy  of  material  actually  oxidized 3,864 

Heat  actually  measured 3,739 


12 


There  is,  therefore,  a  difference  of  only  125  large  calories  in 
this  test,  which  was  one  of  the  early  ones  with  the  new  appa- 
ratus. In  later  experiments  described  by  Atwater  and  his  col- 
leagues much  closer  results  have  been  reported,  which  shows 
the  general  correctness  of  the  method  of  calculation  followed. 
Effect  of  Work.  To  maintain  the  individual  at  work  a 
greater  expenditure  of  energ}^  is  necessary,  and  the  total  en- 
ergy of  substances  metabolized  must  be  balanced  by  the  heat 


THE    ENERGY    EQUATION.  4O I 

liberated  and  external  work  done.  In  this  connection  it  may 
be  well  to  recall  some  relations  first  pointed  out  by  Hirn,  in 
which  a  comparison  is  drawn  between  the  work  of  man  and 
an  engine.  In  both  cases  the  work  is  accomplished  through 
the  expenditure  of  the  potential  energy  stored  up  in  carbonace- 
ous substances,  food  in  the  one  instance,  coal  in  the  other.  As 
the  illustrations  above  show  the  heat  from  the  food  is  prac- 
tically constant,  whether  it  be  evolved  through  oxidation  in 
the  body  or  in  a  calorimeter.  Imagine  now  a  small  steam 
engine  burning  a  constant  amount  of  coal  inside  a  calorimeter. 
In  one  case  let  no  work  be  done  by  the  piston ;  the  heat  of  the 
steam  is  not  employed  in  expansion,  but  is  totally  absorbed  by 
the  water  of  the  calorimeter,  which  takes  up  a  certain  number 
of  calories  that  may  be  accurately  noted.  In  a  second  case 
allow  the  same  amount  of  coal  to  be  burned  under  the  boiler 
of  the  small  engine  in  the  same  time,  but  let  the  engine  do 
work  outside  the  calorimeter  which  may  be  accomplished,  for 
example,  by  means  of  a  small  shaft  passing  through  the  walls 
of  the  calorimeter  in  such  a  way  as  to  convey  no  appreciable 
amount  of  heat.  It  will  now  be  found  that  the  gain  in  tem- 
perature in  the  water  of  the  calorimeter  is  less  than  before 
for  the  same  coal  consumption,  and  that  the  difference  is  meas- 
ured by  the  external  work  alone.  One  calorie  less  in  the 
calorimeter  heat  corresponds  to  423.5  gram-meters  of  work 
done  through  the  agency  of  the  shaft. 

With  an  animal  the  case  is  different.  The  doing  of  exter- 
nal work  necessitates  always  the  burning  of  more  food  than  is 
the  case  with  the  fasting  metabolism,  when  the  energy  require- 
ment is  for  doing  internal  work,  as  will  be  explained  below. 
In  the  engine  the  amount  of  coal  burned  may  be  constant 
whether  work  is  done  or  not.  However,  with  the  animal  this 
result  is  noticed :  While  at  rest  assume  that  for  every  liter  of 
oxygen  absorbed  5  large  calories  of  heat  are  liberated ;  it  does 
not  follow  that  when  work  is  done  and  2  liters  of  oxygen  are 
absorbed  in  the  same  time  that  now  10  Cal.  of  heat  will  be 
liberated.  From  the  10  Cal.  we  must  subtract  the  heat  equiva- 
27 


402  PHYSIOLOGICAL    CHEMISTRY. 

lent  of  the  accomplished  work.  A  part  of  the  increased  heat 
liberation  is  called  for  by  increased  internal  work  also.  It 
follows,  therefore,  that  the  working  animal  is  warmer  than 
the  passive  animal,  but  the  increase  is  not  proportional  to  the 
food  consumed  or  oxyg-en  absorbed. 

External  Work  Equivalent.  Although  the  animal  is  able 
to  convert  but  a  limited  portion  of  the  potential  energy  of  the 
food  into  external  work,  as  a  machine  it  is  still  much  more 
perfect  than  the  steam  engine.  This  is  especially  true  of  man. 
In  the  best  steam  engines  not  more  than  about  12  per  cent  of 
the  potential  energy-  of  the  fuel  can  be  recovered  in  the  form 
of  work.  In  animals,  through  a  short  period,  the  transforma- 
tion may  amount  to  as  much  as  35  per  cent  of  the  net  available 
potential  energy^  In  making  such  comparisons,  however,  it 
must  be  remembered  that  the  animal  can  work  but  a  limited 
time.  In  the  rest  periods  of  the  animal  the  loss  of  heat,  with- 
out any  corresponding  mechanical  gain,  goes  on. 

The  law  defining  the  maximum  conversion  of  heat  into  work  through 
the  steam  engine  is  well  known.  The  extent  of  the  limitations  in  the 
animal  are  not  known.  In  the  steam  engine  the  limitation  depends  on 
the  relation  of  the  highest  heat  of  the  steam  to  the  temperature  of  the 
condenser.  If  T  is  the  absolute  temperature  of  the  live  steam  and  t  the 
temperature  of  the  condenser  the  maximum  transformation  of  heat  into 
work  cannot  be  greater  than 

T 

THE  INTERNAL  WORK  OF   THE  ANIMAL. 

Even  when  the  animal  appears  passive  a  great  deal  of  work 
is  going  on  which  may  be  described  as  internal  work.  The 
nature  and  extent  of  some  of  this  is  known  with  a  fair  degree 
of  accuracy,  while  for  the  extent  of  the  metabolism  correspond- 
ing to  other  kinds  of  work  we  have  not  much  beyond  conjec- 
ture. It  is  possible  to  calculate  approximately  the  work  done 
in  maintaining  the  circulation  of  the  blood,  and  in  respiration, 
and  some  attempts  have  been  made  to  estimate  the  work  of  the 
other  muscles  at  rest;  but  to  approximate  the  work  done  in 
masticating,  digesting,  transporting  and  transforming  the  food 


THE    ENERGY    EQUATION.  4O3 

Stuffs  is  much  more  difficult.  The  work  of  the  heart  alone 
has  been  estimated  at  from  20,000  to  60,000  kilogram-meters 
in  24  hours.  3,000  Cal.  of  heat  liberated  would  correspond 
to  1,270,500  kilogram-meters  of  work;  hence  the  heart  work 
in  forcing  the  blood  through  the  vessels  may  amount  to  as 
much  as  5  per  cent  of  the  whole  metabolism. 

In  respiration  the  work  done  is  largely  the  expansion  of  the 
thorax  against  the  atmospheric  pressure  and  the  elastic  ten- 
sion of  the  rib  cartilages  and  the  lungs.  The  conditions  for 
estimation  are  perhaps  more  favorable  than  in  the  other  case. 
It  has  been  calculated  that  4  to  5  per  cent  of  the  whole  metab- 
olism is  called  for  by  this  work. 

In  maintaining  the  tonus  of  the  great  mass  of  skeletal  mus- 
cles of  the  body  it  is  likely  that  a  large  metabolism  is  required. 
The  muscular  part  of  the  body  is  not  far  from  40  per  cent 
in  the  mean  or  10  per  cent  of  drv^  substance.  In  the  body  of 
a  man  weighing  75  kilograms  we  have  therefore  about  7.5 
kilograms  of  muscle  substance.  A  large  fraction  of  this  falls 
to  the  so-called  skeletal  portion  which  exists  always  in  a  pecu- 
liar tense  condition.  In  keeping  up  this  condition  without 
doing  outside  work  oxidation  is  necessary.  Glycogen  is  split 
up  and  water  and  carbon  dioxide  appear.  The  only  visible 
effect  of  this  metabolism  is  the  production  of  heat.  When  the 
muscle  does  outside  work,  as  in  lifting  a  weight,  although  the 
heat  production  may  be  greater  in  the  sum,  it  is  relatively 
less  in  proportion  to  the  oxygen  consumption.  A  part  of  the 
energy  is  consumed  in  lifting  the  weight. 

Heat  Production  Incidental.  It  is  possible  that  the  whole 
heat  liberation  at  times  is  but  a  result  of  the  A^arious  kinds  of 
internal  work  done,  and  that  no  oxidation  takes  place  for  the 
simple  production  of  heat.  This  may  be  the  case  at  relatively 
high  temperatures.  At  certain  lower  temperatures,  on  the 
other  hand,  it  is  apparent  that  a  part  of  the  heat  liberation  is 
called  for  independently  of  that  transformed  in  the  internal 
work.  A  certain  heat  production  is  necessarily  connected 
with  the  performance  of  the  various  body  functions  and  this 


404  PHYSIOLOGICAL    CHEMISTRY. 

down  to  some  particular  external  temperature  limit  is  suffi- 
cient for  the  heat  demands  of  the  body.  Numerous  experi- 
ments have  shown  that  for  each  animal  species  there  is  an 
external  temperature  at  which  the  general  metabolism,  as  indi- 
cated by  carbon  dioxide  excretion  or  oxygen  consumption,  and 
the  resultant  heat  liberation,  reach  a  minimum  value.  This 
temperature  limit  has  been  called  the  critical  temperature; 
below  it  the  metabolism  and  heat  liberation  increase,  evidently 
not  in  consequence  of  a  call  for  more  work  but  because  of  the 
necessity  for  more  heat. 

ISODYNAMIC    RATIOS. 

In  metabolism,  fats,  carbohydrates  and  proteins  all  yield 
kinetic  energy  and  to  a  large  extent  each  one  is  capable  of 
replacing  the  others,  a  certain  minimum  of  protein  being  al- 
ways, of  course,  necessarily  present.  The  proportions  in 
which  they  may  replace  each  other  may  be  found  by  a  variety 
of  experimental  methods,  which  yield  fairly  concordant  results. 
The  foods  may  be  burned  in  a  calorimeter  and  the  heats  of 
combustion  noted,  or  their  values  may  be  compared  through 
the  amounts  of  oxygen  required  by  calculation  to  oxidize 
them,  or  finally  animal  experiments  in  the  respiration  calorim- 
eter may  be  resorted  to  to  fix  the  relative  values.  The  isody- 
namic  relations  are  given  in  this  table  calculated  from  results 
of  animal  experiments. 

100  gm.  of  fat  = 

Lean  meat,  dry   243  gm. 

Cane  sugar   234  gm. 

Glucose    256  gm. 

Starcli    232  gm. 

For  such  substances  the  calculated  and  observed  values,  or 
the  combustion  calorimeter  and  the  respiration  calorimeter, 
give  closely  agreeing  results,  but  it  must  be  remembered  that 
many  compounds  which  show  considerable  value  as  measured 
by  combustion  are  absolutely  worthless  as  measured  by  nutri- 
tion.    Creatine  and  urea  are  illustrations ;  both  are  products 


THE    ENERGY    EQUATION.  405 

of  metabolism.  On  the  other  hand,  alcohol  shows  about  the 
same  value  in  the  respiration  calorimeter  as  it  shows  in  the 
combustion  calorimeter,  and  its  metabolic  value  would  there- 
fore appear  high.  However  the  actual  food  value  of  alcohol 
is  practically  low  because  limited  by  its  toxic  action. 

FOOD   CONSUMPTION  IN   SEVERE   MUSCULAR   EXERTION. 

In  the  last  chapter  reference  was  made  to  earlier  discussions 
on  the  question  of  the  kind  of  food  which  must  be  metabolized 
to  enable  the  animal  organism  to  do  work.  By  many  authori- 
ties heat  liheration  was  looked  upon  as  an  end  in  itself,  and 
hence  foods  were  divided  into  the  two  classes :  those  important 
in  the  production  of  heat  and  those  important  in  the  produc- 
tion of  outside  work.  The  fats  and  carbohydrates  are  found 
in  the  first  group  and  the  proteins  in  the  second.  One  of  the 
earliest  experimental  investigations  on  the  subject  was  the 
classic  one  of  Fick  and  Wislicenus,  already  referred  to.  These 
two  men  in  1866  made  the  ascent  of  the  Faulhorn  in  the  Swiss 
Alps  from  a  known  level  and  determined  the  excretion  of 
nitrogen,  as  urea,  during  and  following  the  ascent.  As  the 
elevation  to  which  they  ascended  was  known,  it  was  possible 
to  make  some  calculations,  approximately  accurate,  of  the 
work  done  in  the  ascent.  Two  things  were  shown  especially 
by  the  tests  and  calculations :  there  w^as  not  a  great  increase 
in  the  urea  excreted,  and  secondly  the  protein  metabolized,  as 
indicated  by  the  urea  measured,  was  not  at  all  sufficient  to 
account  for  the  work  done.  Their  own  conclusions  were  that 
the  protein  consumed  would  not  furnish  more  than  half  or 
three-fourths  the  energy  necessary  to  lift  their  bodies  through 
the  1956  meters  of  ascent,  to  say  nothing  of  the  work  done  in 
the  horizontal  direction  on  a  winding  pathway,  or  of  the  inter- 
nal work  of  the  body.  This  experiment  attracted  a  great  deal 
of  attention.  Frankland  made  a  new  determination  of  the 
heat  of  combustion  of  protein  and  showed  that  the  value  as- 
sumed by  Fick  and  Wislicenus  was  far  too  high,  thus  making 
the  discrepancy  still  greater. 


4o6 


PHYSIOLOGICAL    CHEMISTRY, 


Since  then  many  similar  observations  have  been  made  which 
show  pretty  clearly  that  when  fats  or  carbohydrates  are  abund- 
ant in  the  food  there  is  no  excessive  destruction  of  protein  in 
the  performance  of  ordinary  work.  With  the  ingestion  of  a 
small  amount  of  protein  it  is  easy  to  cover  the  normal  metab- 
olism. But  the  case  is  different  when  the  work  is  hard. 
Here,  even  with  abundant  iood  and  plenty  of  protein,  there 
appears  to  be  some  loss  of  nitrogen  by  the  body.  In  other 
words,  more  tissue  is  broken  down  than  is  formed  new.  This 
is  brought  out  clearly  in  the  observations  of  Atwater  on  the 
work  done  by  bicyclers  in  a  six-day  race  some  years  ago.  in 
which  the  food  consumed  and  nitrogen  eliminated  were  care- 
fully watched.  The  following  table  gives  a  summary  of  the 
most  important  observations,  with  the  energy  in  large  calories : 


J 
2  ^ 

Protein  Daily. 

] 

energy  Daily 

_u 

■c 

S 

•d 

Rider. 

Average 
per  D 

In  Tota 
Food. 
Grams. 

Availa 

Food. 

Grams. 

■c  E 
^0 

i3  — 

P 

1  >s         1 

s 

s 

A. 

334-6 

169 

158 

223 

4,957 

4,547 

4,789 

B. 

3038 

179 

163  1 

223 

6,300 

5,«7i 

6,066 

C. 

287.7 

211 

»97      1 

243 

4,898 

4,323 

4,464 

Riders  A  and  B  rode  through  six  days.  C  rode  three  days. 
The  energy  metabolized  does  not  include  that  from  body  fat, 
which  may  have  been  considerable. 


DIETARIES. 

The  question  of  the  proper  amount  of  food  and  the  charac- 
ter of  the  food  for  different  kinds  of  work  has  been  very 
thoroughly  studied  in  the  past  few  years  and  a  large  number 
of  observations  on  individuals,  families  and  communities  of 
soldiers,  prisoners  and  paupers  have  been  collected.  The  diet 
in  some  cases  is  known  to  be  sufficient,  in  others  insufficient. 
From  present  experience  it  is  possible  to  say  of  many  dietaries 
that  they  are  excessive,  and  probably  objectionable  in  conse- 


THE   ENERGY   EQUATION. 


407 


quence.  The  following  table  illustrates  the  dietaries  of  a 
great  many  people  living  under  different  conditions.  The  fig- 
ures are  taken  mainly  from  the  compilations  of  Konig  and 
Atwater : 


Occupation,  Etc. 


0.2 


Italian  laborers,  Chicago 4 

Bohemian  laborers,  Chicago 1     8 

Russian  laborers,  Chicago |     9 

Laborers,  crowded  district.  New  York 19 

Laborers,  low  income,  Pittsburgh 

Mechanics,  eastern  and  central  U.  S 

French  Canadians,  Chicago 

French  Canadians,  Massachusetts 

American  professional  men 

Bavarian  workmen,  high  class I     3 

Munich  prisons,  work 

Munich  prisons,  no  work 

Bavarian  soldiers,  war 

Bavarian  soldiers,  garrison 


Protein. 
Grams. 


103 

"5 

137 
106 
81 
103 
118 
III 
105 

151 
104 

87 
145 
120 


Fat. 
Grams. 


Ill 
103 
102 
117 

97 
150 

i5§ 
193 
124 

54 
38 
22 
100 
56 


Carbohy- 
drates, 
Grams. 


391 
360 
418 
367 

3" 

402 

345 
485 
42b 

479 
521 
305 
500 
500 


Calories. 


3,060 
2,885 
3,232 

3>030 
2,510 

3,465 
3.365 
4,23s 
3,335 
3,085 
2,916 
1,819 

3,575 
3,063 


The  above  results  are  fairly  representative  and  show  that 
in  general  over  3000  Cal.  per  day  must  be  provided  in  the 
food.  In  the  figures  the  available  or  net  calories  are  calcu- 
lated from  I  gm.  protein  or  carbohydrate  =4.1  Cal.,  i  gm. 
fat  =  9.3  Cal.  But  many  extreme  results  are  also  found  in 
the  literature.  For  prisoners  confined  in  cells  and  not  work- 
ing, for  paupers  in  asylums,  and  even  with  laborers  poorly 
paid,  the  foods  consumed  may  not  yield  1500  Cal.  On  the 
other  hand,  workmen  in  the  American  winter  lumber  camps, 
who  as  a  rule  are  well  paid,  workmen  in  the  building  trades 
on  outside  work  in  the  colder  weather,  teamsters  and  car 
drivers  who  are  constantly  exposed  to  the  weather,  even  when 
the  work  is  not  excessive,  may  consume  a  diet  yielding  over 
4000  or  even  5000  Cal. 

Special  Diets.  With  such  facts  as  the  above  in  mind  it  is 
not  difficult  to  understand  why  nutrition  with  a  single  article 
of  food  is  unsatisfactory.  Assume,  for  example,  the  case  of 
a  diet  of  potatoes  of  which  the  edible  portion  shows  in  the 


408  PHYSIOLOGICAL    CHEMISTRY. 

mean  alx)iit  these  per  cent  values:  protein  2.2,  fat  o.i.  carbo- 
hydrates 18.4.  100  grams  of  potatoes  would  yield  then  the 
following : 

Protein   2.2  gm.  9.0  Cal. 

Fat    0.1  0.9 

Carbohydrate    18.4  754 

A  pound  of  potatoes  would  furnish,  therefore,  386  Cal.,  and 
6  to  8  pounds  would  have  to  be  consumed  to  furnish  energy 
for  ordinary  work.  The  protein  in  this  would  be  considerably 
below  the  amount  which  has  generally  been  held  necessary, 
while  the  fat  i^  scarcely  appreciable.  The  storage  of  energy 
on  such  a  diet  would  be  practically  impossible. 

On  the  other  hand,  a  diet  of  lean  meat,  round  steak  for 
example,  would  be  almost  as  bad.  This  averages  about  20.9 
per  cent  of  protein  and  10.6  per  cent  of  fat,  from  which  100 
gframs  would  furnish 


& 


Protein   20.9  gm.  85.7  Cal. 

Fat   10.6  98.6 

184.3 

A  pound  would  furnish  835  Cal.  and  about  3  to  4  pounds  would 
have  to  be  consumed  for  support  of  the  body  daily.  While 
the  fat  in  this  would  be  proper  the  protein  would  amount  to 
275  grams  at  least,  and  make  the  work  of  excretion  extremely 
difficult. 

As  a  third  case  a  diet  of  the  small  white  beans  may  be  con- 
sidered. We  have  here  protein  22.5,  fat  1.8,  carbohydrate 
59.6.     In  100  grams,  therefore, 

Protein   22.5  gm.  92.2  Cal. 

Fat   1.8  16.7 

Carbohydrate    59.6  244.3 

353-2 

A  pound  would  furnish  about  1600  Cal.  and  2  pounds  would 
cover  the  needs  of  the  body  practically.     The  proteins  in  this 


THE    ENERGY    EQUATION.  4O9 

would  be  but  slightly  excessive,  while  the  same  would  be  true 
of  carbohydrates.  The  trouble  is  with  the  deficiency  in  fat. 
Notice  how  easily  this  may  be  corrected.  A  pound  and  a  half 
of  beans  cooked  with  one-fourth  pound  of  fat  pork  will  yield 
over  3000  Cal.  and  furnish  a  diet  easily  assimilated  by  men  at 
moderate  work.  What  is  said  of  the  bean  is  practically  true 
of  the  pea;  each  one  approaches  in  value  a  mixed  meat  and 
cereal  diet. 

Required  Protein.  A  much  debated  question  is  that  of 
the  actual  protein  recj[uirement,  supposing  the  other  food  ele- 
ments sufficiently  abundant.  The  standards  given  above  have 
not  gone  unchallenged.  Several  observers  have  described 
metabolism  tests  in  which  less  than  one-half  the  120  or  more 
grams  of  protein,  usually  considered  necessary  in  the  daily 
food,  appears  to  be  sufficient  for  all  needs  and  able  to  maintain 
the  body  in  nitrogen  equilibrium.  Most  of  these  experiments 
have  been,  however,  too  short  to  really  prove  much  definitely. 

But  recently  very  elaborate  and  long  continued  investiga- 
tions on  groups  of  men  have  been  described  by  Chittenden  in 
which  the  evidence  in  favor  of  a  low  protein  requirement  is 
put  in  an  entirely  new  light,  and  in  which  it  is  also  shown  that 
the  3000  large  calories  of  energy  in  our  food  is  more  than 
necessary  for  ordinary  practical  needs.  In  a  group  of  five 
professional  men  Chittenden  found  an  average  nitrogen  libera- 
tion corresponding  to  the  metabolism  of  something  over  46 
gm.  of  protein  daily  and  an  average  energy  value  in  the  whole 
food  of  about  2300  calories  through  a  period  of  six  to  nine 
months.  In  a  group  of  thirteen  soldiers,  taking  abundant  ex- 
ercise, there  was  a  daily  consumption  of  food  having  an  aver- 
age value  of  about  2600  calories,  and  an  average  protein  con- 
sumption of  about  56  gm.  through  five  months,  fall,  winter 
and  early  spring.  In  a  group  of  seven  student  athletes  a  pro- 
tein consumption  of  about  61.5  gm.  daily  with  a  total  food 
consumption  equivalent  to  about  2575  calories  was  observed. 

In  all  these  cases  the  tests  were  continued  long  enough  to 
bring  the  men  into  practical  nitrogen  equilibrium  with  good 


410  PHYSIOLOGICAL    CHEMISTRY. 

physical  condition  and  good  general  health.  For  this  reason 
thev  deserve  the  fullest  attention  and  study.  It  is  the  opinion 
of  the  author  of  the  experiments  that  increased  food  consump- 
tion, so  far  from  being  necessary,  is  even  in  most  cases  a  detri- 
ment, since  it  calls  for  a  large  amount  of  extra  internal  work, 
in  the  liver  and  kidneys  especially,  in  metabolizing  the  diges- 
tion products  and  in  removing  the  waste.  This  is  certainly  a 
consideration  of  some  moment.  How  far  these  findings  may 
be  applied  to  the  case  of  men  at  hard  work,  in  the  open,  in  cold 
weather  remains  to  be  tried.  Chittenden's  results  are  espe- 
cially interesting  with  respect  to  the  necessary  nitrogen ;  but 
for  the  hard-working  man  probably  more  fat  and  more  carbo- 
hydrate will  always  be  found  desirable. 


NDEX. 


Abrin,  265 

Absorption  analysis,  235 

cells,  243 

power,  148 

ratio,  242 
Acetic  acid,  51 

fermentation,  137 
Acetylene  hemoglobin,  226 
Achroodextrin,  47 
Acid,  acetic,  51 

amino-acetic,   "](> 
caproic,    76 
glutaric,   "jd 
isobutylacetic,  76 
propionic,  TJ 
succinic,  76 
valeric,  T] 

arable,  48 

arabonic,  23 

arachidic,  52 

aspartic,  76 

behenic,  52 

butyric,  51,  116,  142 

capric,  52 

caproic,  51 

caprylic,  52 

carbonic,  Tj 

carnic,  181 

cholalic,  320 

cholanic,  320 

choleic,  320 

chondroitin  sulphuric,  107 

cresyl  sulphuric,  358 

dextronic,  23 

elaidic,  58 

erythritic,  23 

feilic,  320 

formic,  51 

glutaminic,  76 

glyceric,  23 

glycerophosphoric,  61 

glycocholic,  320 

glycollic,  23 

hypogaeic,  52 

indoxyl  sulphuric,  358 

lactic,  116 

in  stomach,  161 

lauric,  52 

linoleic,  52 

lithofellic,  321 

mannonic,  23 


Acid,  margaric,  52 

myristic,  52 

nucleinic,  loi,  103 

oenanthylic,  51 

oleic,  58 

oxalic,  23 

oxyphen}d   amino  propionic,   76 

oxyproteic,  361 

palmitic,  52 

parabanic,  I'jj, 

pelargonic,  52 

pentoic,  51 

phenyl  amino  propionic,  76 

phenyl  sulphuric,  358 

phosphoric,  urinary,  359 

picric,  72 

propionic,  51 

pyrrolidine  carboxylic,  '/'j 

ricinoleic,   52 

saccharic,  23 

sarcolactic,  142 

skatoxyl  sulphuric,  358 

stearic,  52 

tartaric,  23 

tartronic,  23 

taurocholic,  321 

thiolactic,   TJ 

trioxyglutaric,  23 

undecylic,  52 

uric,  317,  363 

valeric,  51 
Acid  albumin,  90,  92 
Acid  fermentation  in  intestines,  194 
Acids  in  stomach,  158,  171 

organic  in  stomach,  160 

saturated,  51 
Acrose,  24 
Active  substances,  40 
Addiment,  246 
Adenine,  104,  365 
Adipocere,  57 
Adrenalin,  330 
Agarose,  35 

Agents  of  gastric  digestion,  151 
Agglutinins,  271 
Air,  16 

expired,  17 

tests,   17 
Alanine,  'JJ 

Alanylglycylglycine,  182 
Albumins,  79 


411 


412 


INDEX. 


Albumin,  egg,  8i 

heats  of  coinbustion,  395 

milk.  81 

native,  66 

rotation  of,  81 

serum,  80 
Albuminates,   91 
Albuminoids,  66,  107 
All)umoids,  66 
Albumoses,  93,  95,  178 

in  feces,  210 

tests,  156 
Alcoholic  fermentation,  134 
Alcohol,  tests  for,  136 
Aldehyde  bodies,  22 
Aldohexoses.  23 

Alizarin  sodium  sulphonate,  167 
Alkali  albumin.  90 
Alkalies  and  protein,  75 
Alkaline  fermentation  of  urine,  133 
Alkaloid  reagents,  68 
Alloxan,  373 
Amboceptor,  276 
American  meat  extract,  345 
Amino-acetic  acid,  76 

caproic  acid,  76 

glutaric  acid,  76 

propionic  acid,  T^ 

purine,  365 

succinic  acid,  76 

valeric  acid,  ^^ 
Ammonia,  ^J 

in  urine,  361 

in  water,   13 
Ammoniacal  copper  solution,  37 
Ammonium    carbonate    from    urea, 

132 
Amniotic  fluid,  283 
Amount  of  blood,  214 
Amphopeptone,  97,  81 
Amygdalin,  127 
Amylan,  123 
Amylodextrin,  47 
Amyloid,  66,  III 
Amylopsin,  123,  186 
Amyloses,  42 
Animal  starch,  45 
Animals  and  plants,  relations,  2 
Anti  bodies,  266 

chemical  nature  of,  272 
origin,  273 
Anti  group,  95,  181 
Antitoxins,  266 
Apparatus,  respiration,  382 
Apple  pulp,  darkening  of,  138 
Arabic  acid,  48 
Arabinose,  25 


Arabitols,  2^ 
Arabonic  acid,  2Z 
Arachidic  acid,  52 
Arbacion,  103 
Arginine,  75.  182,  183 
Argon  in  air,   16 
Arrowroot,  43 
Ascites,  282 
Ash  of  bone,  348 

of  milk,  292 
Aspartic  acid,   76 
Asses'  milk,  301 
Assimilation  limit,  368 

of   fats.   204 
Asymmetry.  275 
Atwater  calorimeter,  398 
Autodigestion,  310 
Autolyses,  310 
Average  composition  of  feces,  199 

foods.  112,  113 

milk,  286 

urine,  354 

Bacillus  acidificans  longissimus,  141 

acidi  lactici,  140 
Bacteria,  butyric,  140 

in  feces,  198 

purification  of  water  by,  12 
Bacterial  oxidations,  133 

processes,    192 

reactions,  122 
Bactericidal  action  in  blood,  264 

products  in  autolysis.  313 
Bacteriolytic  processes,  140 
Bacteriolysins,  268 
Bacterium  aceti,  137 
Bases,   21 

hexone,  75 

in  body,  21 
Beans  as  diet,  408 
Beckmann  apparatus,  252 
Beef,  food  value,  112 
Beef  tallow  crystals,  55 
Beet  sugar,  32 

raffinose  in,  35 
Behenic  acid.  52 
Berzelius,  work  of,  5 
Bile,  318 

acids,  optical  properties,  322 
preparation,  321 

analyses,  319 

behavior,  188 

concretions,  327 

functions,  325 

mucin  in,  326 

pigments,  323 
Bilirubin,  229,  323 


INDEX. 


413 


Bilirubin  preparation,  324 
Biliverdin,  229,  323 
Bitch's  milk,  301 
Bitter  almonds,  127 
Biuret  test,  70 
Blood,  213 

analysis,  215 

carbon  dioxide  in,  232 

cholesterol  in,  233 

coagulation,  216 

composition  of,  214 

fibrin  in,  216 

freezing  point,  251 

gases  of,  231 

in   disease,  233 

in  feces,  212 

optical  properties  of,  234 

origin  of,  213 

proteins,  229 

reaction,  219 

salts  of,  231 

self  preservation,  264 

sugar  in,  230 

tests,  218,  219 

variations  in,  214 
Body,  composition  of,  9 

fats,  56 
Bomb  calorimeter,  394 
Bone,  346 

ash,  348  _ 

composition,  18 

marrovi^,  348 
Brain  tissue,  335 
Bran,  pentose  from,  25 
Bread,  leavening,  113 
British  gum,  45 
Bromine  absorption,  61 
Brunner's  glands,    190 
Buchner's  work  on  ferments,  119 
Burchard-Liebermann  reaction,  6s 
Butter,  59 

composition  of,  59 

food  value,  112 

milk,  293 
Butyric  acid,  51,  116 

fermentation,  142 
ferments,  140 

Calcium  compounds  in  urine,  357 

in  body,  10 

lactate,  141 
Calculation  of  kinetic  energy,  396 
Calorie,  392 
Calories  in  food,  112,  113 

required  in  foods,  407,  409 
Calorimeter,  394 
Cane  sugar  group,  32 


Cane  sugar,  specific  rotation,  41 
Capric  acid,  52 
Caproic  acid,  51 
Caprylic  acid,  52 
Carbohydrates,  22 

digestion,  185 

fermentation  of,  123 

in  feces,  206 

in  urine,  368 
Carbonates,  20 

in  water,  20 
Carbon  balance,  381 
Carbon  dioxide  exhaled,  17 
in  air,  16 
in  blood,  232 
Carbon  in  body,  9 
Carbonic  acid  in  body,  20 
Carbon  monoxide  hemoglobin,  224 

poisoning,  225 
Carnic  acid,  181 
Carniferrin,  181 
Carnin,  339 
Cartilage,  348 
Casein,  87,  129 

in  feces,  210 

in  milk,  290 
Caseoses,  95 
Castor  oil,  52 
Catalytic  action,  118 
Cat  fat,  crystals,  55 
Cat's  milk,  301 
Cell  globulins,  83 
Cell,  conductivity,  261 

osmotic  pressure,  249 
Cells,  in  general,  302 

of  starch,  43 
Cellulase,  124 
Celluloid,  SO 
Cellulose,  48 

in  feces,  208 
Cerebrin,  335 
Cerebro  spinal  liquid,  335 
Cetyl  palmitate,  62 
Changes  in  intestines,  192 
Chemical  nature  of  anti  bodies,  272 
Cherry  gum,  25 
Chicken,  food  value,  112 
Chittenden's  dietaries,  409 

pepsin  preparation,  155 
Chlorides,  19 

in  water,  13 
Chlorine  in  body,  10,  19 
Chlorophyll,  3 
Choleic  acid,  320 
Cholesterol,  62 

in  blood,  233 

in  feces,  205 


414 


INDEX. 


Cholesterol  in  gallstones,  2,2^ 

optical  rotation,  328 
Choline,  61 
Cholalic  acid,  320 

in  feces,  205 
Cholanic  acid,  320 
Chondroitin,  107 

sulphuric  acid,  in.  349 
Chondro  mucoid,  107,  349 
Chondrosin,  107 
Chyle,  213,  282 

Classification  of  albumins  and  pep- 
tones. 94 

ferments,  121 

proteins,  66 

sugars,  24 
Cleavage,  hydrolytic,  122 
Clinical  blood  test,  244 
Close  air,  16 
Clupein,   102 

Coagulated  albumins.  89 
Coagulating  proteins,  84 
Coagulation  by  salts,  68 

of  blood,  216 

tests,  67 
Coefficient,  isotonic,  254 
Collagen,  66,  108,  338 
Collodion,  50 
Colors,  bile,  324 
Colostrum,  293 
Combustion,  heat  of,  393 
Commercial  pepsin,   155 
Complement,  276 
Complementoid,  279 
Complete  urine  analyses,  354 
Component  groups  in  proteins,  74 
Composition  of  body.  9 

butter,  59 

urine,  352 
Conductivity  of  blood,  258 

urine.  375 
Configuration,  275 
Congo  red  test,  159 
Conservation  of  energy,  8 
Contents  of  stomach,  test,  158 
Conversion  of  starch,  44 
Copper   solution,   ammoniacal,   37 
Corn  food  value,  113 
Corpuscles,  number  in  blood,  258 
Cow's  milk.  286 
Crackers,  food  value,  113 
Creatine,  338 
Creatinine  in  urine,  367 

reducing  power,  362 
Cresyl  sulphuric  acid,  358 
Cryoscopy,  250 

apparatus,  252 


Cryoscopy  of  urine,  378 
Crystallin,  84 
Crystals,  fat,  55 

hemin,  220 

leucine.  178 

tyrosine,  178 
Cystin,  "]"/,  no 
Cytase,  124 
Cytotoxins,  265,  268 

Dahlia  starch,  45 

Dare's  hemoglobinomctcr.  245,  246 

Denatured  albumins,  90 

Derivatives  of  hemoglobin,  226 

Despretz,  work  of,  7 

Detection  of  acids  in  stomach,  159 

Determination  of  fat,  60 

proteins,  "]}, 

sugars,  35 
Deutero  albumoses,  97 
Dextrins,  44,  47 

specific  rotation,  48 
Dextronic  acid,  2^) 
Diabetes  mellitus,  369 
Dialysis,  82 
Diastase.  117,  123 

behavior  of,  147 
Diazo  reaction,  198 
Dietaries,  406 
Digestion,   115 

carbohydrate,  185 

gastric,   150 

of  starch,  145 

pancreatic,  173 

products  of,  169 
toxicity  of,   184 

salivary,  144 
Diglycylglycine,  182 
Dimethylaminoazobenzene   test,    159 
Dioxyacetone,  23 
Dioxypurine,  365 
Direct  vision  spectroscope,  236 
Disaccharides,  24,  32 
Distearin,  53 
Distillation  of  water,  12 
Distribution  of  food  energj^  395 
Dog's  feces,  normal,  201 
Double  slit,  240 
Dropsy,  282 
Drying  oils.  52 
Dulong,  work  of,  7 
Dyne,  393 

Edestin,  89 
Effects  of  work,  400 
Ehrlich's  theory,  273 
Elaidic  acid,  58 


INDEX. 


415 


Elaidin,  58 

Elastin,  66,  iii 

Electrical  conductivity,  258 

of  urine,  375 
Electrolytes,  258 
Elephant's  milk,  301 
Emulsification,  188 
Emulsin,  117,  127 
Encephalin,  335 
Endogenous  purines,  365 
Endothermal   reactions,   3 
End  products,  351 

of  digestion,  184 
Energy  of  light,  3 

potential,  392 
Enterokinase,  190 
Enzymes,  115 
Epinephrin,  330 
Erepsin,  190 
Erg,  393 

Erythritic  acid,  23 
Erythrodextrin,  47 
Erythrogranulose,  48 
Erythrol,  23 
Erythrose,  2;^ 

Ethereal  sulphates,  316,  358 
Ethers  of  glucose,  127 
Eumycetes,  134 
Ewald's  test  meal,  158 
Examination    of    stomach    contents, 

158    . 
Excretion,  gaseous,  380 

nitrogenous,  351 
Exogenous  purines,  365 
Experiments  in  fermentation,  135 
External  work  equivalent,  402 
Extinction  coefficient,  241 
Extraction  of  liver,  305 

muscle,  336 
Extract  of  malt,  146 

meat,  343 

pancreas,  175 
Exudations,  282 

Factors  for  titration  of  sugar,  36 
Fasting  animals,   respiratory   coeffi- 
cient, 384 
Fat  crystals,  55 

determination  of,  60 

human,  60 

in  milk,  289 

in  muscle,  338 

splitting,  122,  128 
Fats,  51 

digestion,  187 

heat  of  combusion,  395 

in  feces,  203 


Fats,  loss  in  digestion,  204 
Fatty  acids,  51 
Faulhorn  experiment,  383 
Feathers,   no 
Feces,  192 

albumin  in,  210 

analyses  of,  201 

carbohydrates  in,  206 

casein  in,  210 

cholalic  acid  in,  205 

cholesterol  in,  205 

composition,  198 

fats  in,  213 

formation,  196 

from  various  foods,  200 

gums  in,  208 

lecithin  in,  206 

mucus  in,  211 

nitrogen  in,  208 

normal,  199 

nucleoproteids  in,  211 

peptone  in,  210 

reaction,  202 

separation,  201 

soaps  in,  206 

specific  gravity,  203 
Fehling's  solution,  28 
Fellic  acid,  320 
Ferment,  inverting,  126 
Fermentation,  117 

acetic,    137 

alcohol,  134 

autolytic,  310 

butyric,   142 

lactic,  140 

mucous,   143 
Ferments,  115 

bacterial,  140 

specific  action,  275 
Fibrin,  84 

in  blood,  216 
Fibrinogen,  84 
Fibrinoses,  95 
Fick    and    Wislicenus    experiment, 

383 
Filter  paper,  48 
Filtration  of  water,  12 
Finger  nails,  no 
Fish,  food  value,  112 
Fish  scales,  collagin  in,  108 
Fleischl  hemometer,  244 
Flesh  bases,  338 
Flour,  112 

food  value,  113 

tests,   112 
Fluorine  in  body,   10 
Food  consumption,  405 


4i6 


INDEX. 


Food,  effect  on  feces,  200 

energy  of,  392 

fuel  value,  112.  113 

Iieats  of  combustion,  395 

kinetic  energy  from,  396 

nucleins,  365 

of  laborers,  407 

requisites,  408 

stuffs.   III 
Force,  unit  of,  393 
Formaldebyde,  3,  24 
Formic  acid,  51 

Formulas  for  quantitative  spectrum 
analysis,  241,  242 

of  carbohydrates,  22 
Freezing  point  methods,  251 

of  urine,  378 
Fruit  juices,  sugar  in,  2"] 
Fructose,  23.  31 

specific  rotation,  41 
Fundus  glands,  150 
Fungi  budding,  134 

fission,  134 
Furfuraldehyde,  26 
Furoaniliuie,  26 

Galactonic  acid.  31 
Galactose,  31,  109,  125 
Gallstones,  62,  327 
Gaseous  excretion,  380 
Gases  of  blood,  231 
Gastric  juice,  150 

titration,  166 
Gelatin,  108,  346 

and  bacteria,  109 
Glands  of  intestine,  189 

salivary,   144 
Gliadin,  88 
Globin,  103 
Globinoses,  95 
Globulins,  83 
Glucase,  124 
Glucosamine,  7J 
Glucoproteids,  105 
Glucose,  27 

from  starch,  44 

in  muscle,  341 

specific  rotation,  41 
Glucosides,  26,  127 
Glucosone,  30 
Glue,  108 

Glutaminic  acid,  76 
Gluten.  88 
Glutenin,  88 
Glutin,  108 
Glyceric  acid,  23 
Glycerol,  23,  54.  60 


Glycerophosphoric  acid,  61 
Glycerose,  23 
Glycine,  76 
Glj'cocholic  acid,  320 
Glycocoll,  76,  109 
Glycocollic  acid,  23 
Glycogen,  45 

as  reserve  material,  46 

destruction  of,  309 

in  liver,  306 

in  muscle.  339 

specific  rotation,  46 
Gmclin,  work  of,  5 
Goat's  milk.  301 
Gower's  hemoglobinometer,  246 
Graham  dialyzer,  82 
Granulose,  42 

Groups  in  protein  molecule,  74 
Guaiacum  test,  219 
Guanine,  104,  365 
Guenzberg's  reagent,  159 
Gum,  British,  45 
Gums,  47 

in  feces,  208 

sugar  from,  25 

Hair  dye,  no 

Ham,  food  value,   112 

Haptophorous  group,  276 

Hardness  of  water,  11 

Hard  soap,  53 

Heat  and  work,  400 

Heat  energy,  distribution  of,  399 

mechanical  equivalent,  393 

of  combustion,  393 

production,  incidental,  403 

unit,  392 
Hematin,  227,  2,^2> 

spectrum,  239 
Hematocrit,  clinical  uses,  258 

Koeppe's  256 

methods,  255 
Hematogen,  88 
Hematoidin,  229 
Hematoporphyrin,  228,  ^i^z 
Hematolin,  228,  229 
Hemicelluloses,  49 
Hemi  group,  95,  181 
Hemin,  228 

crystals,  220 
Hemachromogen,  228,  229 
Hemoglobin,  105,  221 

analyses,  221 

combinations  of,  222 

crystals,  223 

derivatives,  226  ' 

formulas,  222 


INDEX. 


417 


Hemoglobin,  spectra,  238 

Hemoglobinometer,  245 

Hemolysins,  268 

Hemometer,  244 

Herbivora,  nutrition  by  pentoses,  26 

Heteroalbumose,  97 

Hexitols,  23 

Hexone  bases,  75,  181,  183 

Hexoses,  26 

Histidine,  75,  182,  183 

Histones,  loi 

Historical  notes,  4 

Hog's  pancreas,  ferments,  175 

Hoppe-Seyler,  work  of,  6 

Horn,  350 

Human  fat,  60 

milk,  297 
Hydrazones,  27,  30 
Hydrocele,  282 
Hydrogen  in  body,  9 
Hydrolytic  cleavage,  122 

reactions,  122 
Hydrothorax,  282 
Hypochlorite   reaction,   363 
Hypogasic  acid,  52 
Hypoxanthine,  365 

Ichthulin,  89 

Identity  of  pepsin  and  rennin,  131 

Immune  body,  276 

sera.  268 
Immunization,  270 
Incidental  heat  production,  403 
Indestructibility  of  matter,  8 
'Indican,  179,  196,  359 
Indicators,  163 
Indol,  179 
Indoxyl  sulphate,  179 

sulphuric  acid,  358 
Inorganic  elements,  9 
Inosite,  340 
Intermediary  body,  276 
Internal  work,  402 
Interpretation  of  feces  analyses,  201 
Intestinal  bacteria,   193 

j'uice,  189 
Inulase,  124 
Inulin,  45 

Inversion  of  sugars,  32 
Irivertase,  33,  126,  136 
Invert  sugar,  32 

specific  rotation^  41 
Iodine,  absorption,  61 

and  starch,  44 

in  body,  9 
lodothyreoglobulin,  332 
lodothyrin,  23 

28 


Iron  in  body,  10,  21 
Isinglass,  no 
Isodynamic   ratios,  404 
Isomaltose,  35 
Isotonic  coefficient,  254 

Japanese  lacquer,  139 
Jelly  of  Lieberkiihn,  91 
Joule,  work  on  heat,  7 
Juice,  intestinal,  189 

Kephir,  34,  142 
Kelling's  test,  161 
Keratin,  66,  no,  349 
Ketohexoses,  23 
Ketones,  23 
Kinetic  energy,  395 
Kjeldahl  process,  y^ 
Kohlrausch  apparatus,  262 
Koeppe's  hematocrit,  256 
Kruess'  spectroscope,  240 
Kuehne,  pepsin  preparation,  155 

work  of,  6 
Kumyss,  sSt   ^4~ 

Laccase,  139 

Lacquer,  139 

Lactalbumin,  81,  290 

Lactase,  125 

Lactate,  calcium,  141 
zinc,  141 

Lactic  acid,  116 

in  autolysis,  312 
in  intestines,  194 
in  muscle,  341 
in  stomach,  161 ' 

Lactic  bacteria,  140 

Lactose,  S3^  125 

Lactosazone,  34 

Landwehr's  animal  gum,  106 

Lanolin,  62 

Laurent  polariscope,  39 

Laurie  acid,  52 

Lavoisier's  work,  4 

Lard,  58 

Lead  hydroxide  test,  72 

Lead  plaster,  52,  54 

Lecithin,  61,  303 
in  feces,  206 

Legumin,  89 

Lehmann,  work  of,  6 

Leucine,  76,  in,  177 

Leucylproline,  182 

Levulose,  31 

specific  rotation,  41 

Lieberkiilm's  glands,  189 
jelly,  91 


4i8 


INDEX. 


Licbip's  theory  of  fermentation,  117 

Licbigr,  work  of,  5 

Lignocelluloses,  49 

Linoleic  acid,  52 

Lipase.  128.  187 

Liquid  resistance,  260 

Lithium  in  body,  9 

Lithofellic  acid.  321 

Liver  and  poisons,  314 

and  uric  acid,  317 

cells  composition,  304 

changes  in.  307 

chemistry  of.  302 

fermentation.   306 

glycogen,  46,  306 

syntheses  in,  314 
Loewe's  sohition,  38 
Lymph,  280 

cells,   283 

functions.  281 
Lysine,  75,  182,  183 

Magnesium  compounds  in  urine,  357 

in   body.    10 
Malondiamide,  70 
Malt.  146 

diastase,  34 
Maltase.  124.  185.  186 
Maltose,  34,  146 
Malt  sugar,  145 

uses  of,  34 

specific  rotation,  41 
Mannitol.  23 
Mannonic  acid,  23 
Mare's  milk.  301 
Margaric  acid,  52 
Margarin,  58 
Market  milk,  287 
Marrow,  348 
Mayer,  work  on  heat,  7 
Meal,  112 
Meat  as  diet,  408 

extracts,  343 

food  vaUie,  112 
Melibiose,  35 
Melitose,  35 

Metabolism  experiments,  386 
Methemoglobin,  227 

spectrum,  239 
Methyl  orange,  indicator,  163 

violet  test,  159 
Micoderma  aceti.  137 
Micrococcus  urese.  133 
Microorganisms,   116 
Milk,  114,  286 

analysis  of,  295 

ash,  292 


Milk,  casein  in,  290 

fat  in,  289 

human.  297 

mineral  substances,  291 

modified,  299 
Milk  of  ass,  301 

bitch,  301 

cat.  301 

elephant.  301 

goat,  301 

mare,  301 

sheep,  301 

sow,  30  r 

origin  of,  288 

preservatives,  297 

sugar,  31,  ss,  291 

specific  rotation,  41 
uses  of,  34 
Millon's  reagent,  69 

test,  177 
Mineral  substances,  17 
Mixtures,  conductivity  of,  376 
Modification  of  milk,  299 
Modified  albumins,  89 

milk,  299 
Moisture  in  air,  16 
Molecular  asymmetry,  275 
Molisch  test,  29,  71 
IVIonosaccharides,   24 
Monostearin,  53 
Monoses,  24 
Mother  of  vinegar,   137 
Mother's  milk,  297 
Mucic  acid,  31 
Mucin  in  bile,  326 

in  feces,  211 
Mucins,   IDS 
Mucoids,  105,  106 
Mucor  mucedo,  134 
Mucous  fermentation,   143 
Muscle  ash.  343 

composition  of,  336 

glucose  in,  341 

lactic  acid  in,  341 

oxidation  in.  340 

proteins,  237 
Muscular  exertion  and  food,  405 
Aiusculin,  337 
Mustard  oil,  127 
Mutton  fat  crystals,  55 

food  value,   112 
Mycose,  35 
Myogen.  85,  :^:^7 
Myosin,  85,  337 
Myosinogen,  85 
Myosinoses,  95 
Myricin,  62 


INDEX. 


419 


Myricyl  palmitate,  62 
Myristic  acid,  52 
Myrosin,  127 

Nails,  350 

Naphthol  tests,  70,  71 
Native  albumins,  66,  79 
Natural  fats,  51 
Nerve  substance,  335 
Nessler's  reagent,  13 
Nicol  prism,  39 
Nitrates  in  water,  14 
Nitrates  of  cellulose,  50 
Nitric  oxide  hemoglobin,  226 
Nitrites  in  water,  14 
Nitro  celluloses,  50 
Nitrogen  balance,  381 

in  air,  16 

in  body,  10 

in  feces,  208 

in  urine,  360 

required  in  food,  409 
Nitrogenous  excretion,  351 
Nonsaturated  acids,  52 
Normal  freezing  point  of  blood,  253 

reduction,  371 
Nucleinic  acid,  loi,  103 
Nucleins,  100 
Nucleo  albumins,  86 

histone,   103 

proteids,  100 
in  feces,  211 
Nucleus  of  cells,  303 
Nutritives,  9 
Nutrose,  87 
Nuts,  food  value,  113 

Occurrence  of  starch,  42 
Odor  of  intestine,  193 
CEnanthylic  acid,  51 
Oil,  oleo,  59 
Oleic  acid,  58 
Olein,  58 
Oleomargarin,  59 

food  value,  112 
Optical  properties  of  bile  acids,  322 
of  blood,  234 

rotation  of  sugars,  39 
Organic  acids   formed  in  autolysis, 

311 
in  stomach,  160 
chemistry,     relations    to    phys- 
iology and  pathology,  5 
combinations  of  bases,  21 
Organized  ferments,  119 
Organo-metallic  bodies,  21 
Origin  of  fat  in  body,  56 


Origin     of     hydrochloric     acid     in 

stomach,  151 
Osozones,  27,  30 
Osmotic  pressure,  248 

in  urine,  379 
Osones,  27,  30 
Ossein,  347 

Oxalates  and  blood  coagulation,  217 
Oxalic  acid,  23 

from  starch,  44 
Oxaluramide,  70 
Oxamide,  70 
Oxidases,  138 
Oxidation  fermentations,  133 

place  of,  390 

reactions,  122 

tests  for  water,  13 
Oxygen  absorbed  by  hemoglobin,  224 

in  air,  16 

in  body,  9 

respiration,  385 
Oxyhemoglobin  spectrum,  222,  236 
Oxyproteic  acid,  361 
Oxypurine,  365 
Oysters,  food  value,  112 

Palmitic  acid,  52 

Palmitin,  58 

Pancreas  and  fats,  187 

autolysis,  330 

chemistry  of,  329 

powder,  175 
Pancreatic  digestion,   173 

enzymes,  174 

juice,  173 
Paper,  cellulose  in,  48 
Parabanic  acid,  373 
Para  casein,  87 
Paste,  starch,  44 

Pasteur's  theory  of  fermentation,  118 
Pasteur,  work  of,  6 
Pavy  solution,  38,  372 
Pawlow  school,  131 
Pea  legumin,  89 
Peanut  oil,  52 
Peas,  food  value,  113 
Pectin,  124 

sugar,  25 
Pectinase,  124 
Pelargonic  acid,  52 
Pentitols,  23 
Pentoic  acid,  51 
Pentosans,  25 
Pentose,  23 
Pentoses,  25 
Pepsin,  129,  152 

amount  of  "in  stomach,  167 


420 


INDEX. 


Pepsin,  commercial,  155 
isolation,   154 
-peptone,  183 
preparation,  154 
tests,  156 
valuation,  157 
Pepsinogen.  130 
Peptones,  93,   178 
in  feces.  210 
tests.  96,  156 
Peptonization  of  milk,  294 
Percentage  composition  of  body,  9, 
10 
variation  in  urine,  iS3 
Permanganate  reagent,  14 
Perspiration,  losses  in,  389 
Pfeiffer's  phenomenon,  274 
Phagocytes,  264 
Phenol-phthalem  indicator.  163 
Phenols  formed  in  intestines,  195 
Phenomena  of  Pfeiffer,  274 
Phenyl  alanine.  76 

alanylglycylglycine,   182 
amino  propionic  acid,  76 
glucosazone,  30 
hydrazine,  27 
tests,   30 
sulphuric  acid,  358 
Phloroglucin  test,  159 
Phosphates.  17 

in  urine.  359 
Phosphocarnic  acid,  181 
Phosphorus  in  body,,  10 

in  urine,  359 
Physical  composition  of  milk,  287 

methods,  248 
Physiological   chemistry,   scope,  2 

importance  of  water,  15 
Phytoglobulins,  84 
Phytovitellins.  84 
Picric  acid,  72 
Pigment  tests,  bile,  324 
Pigmentation,  258 
Pigments  of  bile,  323 
Plants  and  animals,  relations,  2 
Plasmon,  87 

Pleural  transudates,  283 
Poisoning  by  carbon  monoxide,  225 
Poisons  and  liver,  314 

formed  in  intestines,  197 
Polariscope,  39 
Polarization  tests,  39 
Polyhydric  alcohols,  23 
Polypeptides,  78,   182 
Polysaccharides,  24,  41 
Potassium  in  body,  10 
indoxyl  sulphate,"  179 


Potassium  permanganate  reagent,  14 
Potatoes  as  diet,  407 

food  value.   113 

starch  from.  43   ' 
Potential  energy  of  food,  392 
Powder,  pancreas,  175 
Precipitation  by  salts,  68 

limits,  67 
Precipitins,  267 
Preparation  of  bile  acid,  321 
Preservatives  of  milk,  297 
Pressure,  osmotic,  248 
Primary  albumoses,  96 

phosphates,  18 
Products  of  peptic  digestion,  169 

tr>-ptic  digestion,  180 
Proline.   182 
Propepsin,   130 
Propionic  acid,  51 
Protagon,  335 
Protamine,  loi 
Proteids.  66,  100 
Protein  coagulation,  68 

requirement,  409 

substances,  64 

synthesis,  78 
Proteins  and  alkalies,  75 

as  bases,  152 

autolysis.  312 

classification.  65 

coagulating,  84 

determination  of,  73 

formulas,  65 

heats  of  combustion,  395 

in  feces,  208 

in  milk,  294 

in  muscle,  337 

reaction  of,  66 
Proteoses,  95 
Proteolytic  reactions,  128 
Prothrombin,  216 
Protones.  102 
Protoplasm,  303 
Pseudo  acids,  69 

bases,  69,  79 

celluloses,  49 

pepsin,  190 
Psychic  stimulus,  150 
Ptyalin,  117,  146 
Purification  of  water,  12 
Purine  bases,  104 

bodies,  363 
Pus  cells,  composition,  284 
in  feces,  212 

serum.  283 
Putrefaction,  192 
Pyloric  glands,  150 


INDEX. 


421 


Pyrimidine,  367 

Pyrrolidine  carboxylic  acid,  yj,  82 

Quadriurates,  366 

Quantitative  spectrum  analysis,  239 

Quince  mucilage,  25 

Quotient,  respiratory,  380 

Radiation  of  heat,  400 
Raffinose,  35 

in  beet  sugar,  35 
Ratios,  isodynamic,  404 
Reaction  of  blood,  219 

of  feces,  202 

xanthoproteic,  '!2 
Reactions,  aldose,  28 

ferment,   121 

of  fats,  52 

pepsin,  156 

sugar,  28 
Receptors,  276 

Reducing  power  of  sugars,  36 
Reduction,  normal  in  urine,  371 
Relations  of  carbohydrates,  23 
Rennet,  129 
Rennin,  87,  129,  152 
Reproductive  glands,  333 
Resistance  capacity,  260 

electrical,  260 
Resorcinol  test,  160 
Respiration  test,  380 

apparatus,  382 

calorimeters,  397 

skin,  388 
Respiratory  quotient  illustration,  385 
Ricin,  265 
Ricinoleic  acid,  52 
Riegel  test  meal,  158 
Rumford,  discoveries,  7 

Saccharic  acid,  23 
Saccharodioses,  24 
Saccharomyces,   134 
Saccharose,  32 
Saccharotrioses,  24,  35 
Salicin,  127 
Saliginin,  127 
Saliva,  144 

alkalinity  of,  148 
Salivary  diastase,  117,  123 
Salkowski's  test,  63 
Salmin,  102 
Salmo  histone,  103 
Salmon,   103 
Salts  in  urine,  355 

of  blood,  231 

of  milk,  292 


Salts,   physiologically  important,   t8 
Sand  filtration,  12 
Saponification,  52 
Sarcolactic  acid,   142 
Saturated  acids,  51 
Schizomycetes,  134 
Schulz  prism,  243 
Schiitzenberger,  v^fork  of,  6 
Schweitzer's  reagent,  49,  50 
Scomber  histone,  103 
Scombrin,  102 
Scombron,  103 
Secondary  albumoses,  96 

phosphates,  18 
Sediment  of  urates,  366 

or  urine,  370 
Self  digestion,  310 

preservation  of  blood,  264 

purification  of  water,  12 

regeneration  of  cells.  278 
Serum  albumin,  80 

globulin,  80,  83 

precipitins,  267 
Sheep's  milk,  301 
Side  chain  theory,  274 
Silicon  in  body,  10 
Size,  45 

Size  of  starch  cells,  43 
Skatol,  179 

Skatoxyl  sulphuric  acid,  358 
Skimmed  milk,  293 
Skin  respiration,  388 
Soaps,   52 

in  feces,  206 
Soda  lime  test,  67 
Sodium  in  body,  10 
Soft  water,  11 
Solids  in  feces,  202 
Soluble  starch,  43 
Sorbinose,  32 
Sour  milk,  acid  in,  116 
Sow's  milk,  301 

Soxhlet's  table  of  reducing  power,  z^ 
Special  diets,  407 
Specific  gravity  of  blood,  214 
feces,  203 
urine,  354 

rotation,  40 
Spectra  of  blood,  237 
Spectrophotometer,  240 
Spectroscopes,  235,  236 
Spectrum  analysis,  239 

of  blood,  234 
Spermatic  fluid,  334 
Spermatozoa,  334 

products  from,  loi 
Spermine,  334 


422 


INDEX. 


Spleen,  284 
Spongin,  6(5 
Standards  of  food,  406 
Starch,  42 

animal,  45 

as  food,  45 

digestion,  124,  145 

in  feces,  206 
Steapsin,  128,  187 
Stearic  acid,  52 
Stearin,  57,  102,  187 

decomposition,  74 
Stimuli  for  digestion,  150 
Stomach  digestion,  150 

function  of,  170 

tests,  153 
Sucrase,  126 

Sucrose,  inversion  of,  32,  126 
Sugar,  in  beets,  32 

blood,  230 

canes,  32 

feces,  206 

malt,  34 

milk,  33 

saps,  32 

of  milk,  291 
Sugars,  determination  of,  35 

heats  of  combustion,  395 

optical  rotation  of,  39 

synthesis  of,  25 

tests,  29 
Sulphates,  ethereal,  316 

in  urine,  20,  358 
Sulpho-hemoglobin,  226 
Sulphur  in  body,  10,  20 

protein,  20,  72,  yo 

urine,  358 
Suprarenal  bodies,  330 
Suprarenin,  330 
Symbiotic  fermentations,  142 
Synthesis  in  plants,  2 

of  polypeptides,  78,  182 

of  sugars,  25 
Synthetic  processes  in  liver,  314 
Syntonine,  92 
Table,  absorption  ratios,  243 

albumoses  and  peptones,  99 

analyses  of  transudate,  283 

bile  analyses,  319 

blood  analyses,  215 

dietaries,  407 

elements  of  body,  9 

heats  of  combustion,  395 

hemoglobin  analyses,  221 

isotonic  coefficients,  255 

milk  salts,  292 

specific  rotation.  41 


Table  of  urine  analyses,  354 
conductivity.  ^Jj 

Tallow.  58 
Tallquist's  chart.  247 
Talose.  32 
Tartaric  acid.  23 
Taurocholic  acid,  321 
Teichmann's  crystals,  220 
Tendon  mucoid,  106 
Tertiary  phosphates,  18 
Test,  bismuth,  29 

biuret,  70 

Burchard-Liebermann,   63 

cholesterol,  63 

coagulation,  67 

Congo,  159 

dimethylaminoazobenzene,  159 

Fehling's,  29 

Gmelin's  bile,  324 

guaiacum,  219 

Guenzberg's,  159 

Hammarsten's  bile,  324 

heat,  for  blood,  220 

hydrogen    peroxide    for    blood, 
219 

iodine,  44 

Kelling's,  161 

lead  hydroxide,  72 

methyl  violet.   159 

Millon,  70,  177 

Molisch,  29,  71 

oxidation,  13 

phenyl  hydrazine,  30 

Salkowski's,  63 

soda  lime,  67 

Trommer's,  29 

Uffelman's,  i6l 

Widal,  271 

xanthoproteic,  72 
Tests  for  air,  17 

albumose,  96,   156 

alcohol,  136 

ammonia  in  water,  13 

bile  acids,  322 

chlorides  in  water,  13 

fat  in  milk,  293 

fats,  53 

gelatin,   109 

glycogen,  46 

leucine,  178 

nitrates,  14 

nitrites,  14 

pepsin,  156 

peptone,  96,  156 

protein  in  milk,  294 

starch,  43 


INDEX. 


423 


Tests,  sugar  in  milk,  294 
stomach  contents,  158 
Test  meals,  158 
Tetrahydroxymono-carboxylic   acid, 

23 
Tetroses,  2^ 

Theories  of  fermentation,  117 
Theory  of  Ehrlich,  273 

indicators,  163 
Thiocyanates  in  saliva,  144 
Thiolactic  acid,  77 
Thompson,  Benjamin,  work  of,  7 
Thrombrin,  216 
Thymine,  367 

Thymus  cells,  analysis  of,  284 
Thyreoglobulin,  332 
Thyroid,  chemistry  of,  332 
Thyroiodine,  333 
Tissue,  oxidation,  390 
Tissues,  water  in,  15 
Titration  of  gastric  juice,  166 

Volhard's,  162 
Total  hydrochloric  acid,  160 
Toxicity  of  crude  albumose,  185 

digestion  products,  184 
Toxins,  268 

in  intestines,  197 
Toxoids,  279 

Transformation  products,  66,  89 
Transfusion  of  blood,  269 
Transudations,  280 
Trehalose,  35 

Trihydroxydicarboxjdic  acid,  23 
Triolein,  58 
Triose,  23 

Trioxyglutaric  acid,  23 
Trioxypurine,   104,  365 
Tripalmitin,  58 
Triple  phosphate,  360 
Trisaccharides,  24,  35 
Tristearin,  57 
Tritico  nucleinic  acid,  104 
Trommer's  test,  29 
True  albumins,  66 
Trypsin,  131,  I74 

antipeptone,  183 
Trypsinogen,  190 
Tryptophane,   176 
Turanose,  35 
Tyrosine,  76,  139,  177 

tests,  70 
Tyrosinase,  139 

Uffelmann's  test,  161 
Undecylic  acid,  52 
Unit  of  heat,  392 
Unorganized  ferments,  119 


Uracil,  104,  367 
Urea,  356 

fermentation,  123 

in  liver,  314 

in  urine,  362 
Urease,  132 
Uric  acid,  356,  363 

in  liver,  317 

reducing  power,  273 
Urine,  351 

analyses,  354 

carbohydrates,  368 

creatinine  in,  367 

electrical  conductivity,  375 

fermentation,   132 

freezing  point,  378 

general  composition,  352 

normal  reducing  power,  371 

proteins  in,  370 

purine  bodies,  365 

salts  in,  357 

sediment,  370 

sulphur  in,  358 

urea,  362 

uric  acid,  363 

Valeric  acid,  51 

Value  of  blood  conductivity,  261 
VanilHn  test,  159 
Variations  in  urine,  353 
Vegetables,  food  value,  113 
nucleo-albumins  in,  88 
Vinegar,  137 
Vitellin,  88 
Voit,  work  of,  6 
Volhard's  titration,  162 

Water,  hardness,  il 

in  body,  11 

physiological  importance,   15 

purification,  12 

tests  for,  12 
Waxes,  62 
Weigert's  doctrine  of  regeneration, 

278 
Wheat  flour  composition,  88,  113 

starch,  42 
Wheatstone  bridge,  260 
Whey,  293 
Widal  test,  271 
Wittich's  pepsin,  154 
Wohler,  work  of,  5 
Wood  cellulose,  49 

sugar,  26 
Work  and  heat,  400 

unit  of,  393 


424  INDEX. 

Xantliine.  365.367  Yeast  and  flour,  113 

bases,  104  enzymes   in,   120 
bodies,  364 

bodies  in  muscle,  339  Zinc  chloride  creatinine,  368 

Xanthoproteic  test,  T2  lactate,  141 

Xylose,  26  Zones  of  fermentation,  194 

Zymase,  120,  136 

Yeast,  134  Zymotoxic  group,  278 


COLUMBIA   UNIVERSITY 

This  book   is  due  on  the  date  indicated  below,  or  at  the 
expiration  of  a  definite  period  after  the  date  of  borrowing, 
as  provided  by  the  rules  of  the  Library  or  by  special  ar- 
rangement with  the  Librarian  in  charge. 

DATE  BORROWED 

DATE  DUE 

DATE  BORROWED 

DATE  DUE 

C2a'63a>M50 

1^5 


Long 


,v  of  piiysiQ^°°^  .,^.^— - 


